Chapter 12:  Intracellular Compartments and Protein Sorting

All Eucaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles

Many vital biochemical processes take place in or on membrane surfaces. Lipid metabolism, for example, is catalyzed mostly by membrane-bound enzymes, and oxidative phosphorylation and photosynthesis both require a membrane to couple the transport of H⁺ to the synthesis of ATP. Intracellular membrane systems, however, do more for the cell than just provide increased membrane area: they create enclosed compartments that are separate from the cytosol, thus providing the cell with functionally specialized aqueous spaces. Because the lipid bilayer of organelle membranes is impermeable to most hydrophilic molecules, the membrane of each organelle must contain membrane transport proteins that are responsible for the import and export of specific metabolites. Each organelle membrane must also have a mechanism for importing, and incorporating into the organelle, the specific proteins that make the organelle unique.

The major intracellular compartments common to eucaryotic cells are illustrated in Figure 12-1. The nucleus contains the main genome and is the principal site of DNA and RNA synthesis. The surrounding cytoplasm consists of the cytosol and the cytoplasmic organelles suspended in it. The cytosol, constituting a little more than half the total volume of the cell, is the site of protein synthesis and degradation. It also performs most of the cell's intermediary metabolism—that is, the many reactions by which some small molecules are degraded and others are synthesized to provide the building blocks for macromolecules (discussed in Chapter 2).

About half the total area of membrane in a eucaryotic cell encloses the labyrinthine spaces of the endoplasmic reticulum (ER). The ER has many ribosomes bound to its cytosolic surface; these are engaged in the synthesis of both soluble and integral membrane proteins, most of which are destined either for secretion to the cell exterior or for other organelles. We shall see that whereas proteins are translocated into other organelles only after their synthesis is complete, they are translocated into the ER as they are synthesized. This explains why the ER membrane is unique in having ribosomes tethered to it. The ER also produces most of the lipid for the rest of the cell and functions as a store for Ca²⁺ ions. The ER sends many of its proteins and lipids to the Golgi apparatus. The Golgi apparatus consists of organized stacks of disclike compartments called Golgi cisternae; it receives lipids and proteins from the ER and dispatches them to a variety of destinations, usually covalently modifying them en route.

Mitochondria and (in plants) chloroplasts generate most of the ATP used by cells to drive reactions that require an input of free energy; chloroplasts are a specialized version of plastids, which can also have other functions in plant cells, such as the storage of food or pigment molecules. Lysosomes contain digestive enzymes that degrade defunct intracellular organelles, as well as macromolecules and particles taken in from outside the cell by endocytosis. On their way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes. Peroxisomes are small vesicular compartments that contain enzymes utilized in a variety of oxidative reactions.

In general, each membrane-enclosed organelle performs the same set of basic functions in all cell types. But to serve the specialized functions of cells, these organelles will vary in abundance and can have additional properties that differ from cell type to cell type.

On average, the membrane-enclosed compartments together occupy nearly half the volume of a cell (Table 12-1), and a large amount of intracellular membrane is required to make them all. In liver and pancreatic cells, for example, the endoplasmic reticulum has a total membrane surface area that is, respectively, 25 times and 12 times that of the plasma membrane (Table 12-2). In terms of its area and mass, the plasma membrane is only a minor membrane in most eucaryotic cells (Figure 12-2).

Membrane-enclosed organelles often have characteristic positions in the cytosol. In most cells, for example, the Golgi apparatus is located close to the nucleus, whereas the network of ER tubules extends from the nucleus throughout the entire cytosol. These characteristic distributions depend on interactions of the organelles with the cytoskeleton. The localization of both the ER and the Golgi apparatus, for instance, depends on an intact microtubule array; if the microtubules are experimentally depolymerized with a drug, the Golgi apparatus fragments and disperses throughout the cell, and the ER network collapses toward the cell center (discussed in Chapter 16).

The Topological Relationships of Membrane-enclosed Organelles Can Be Interpreted in Terms of Their Evolutionary Origins

To understand the relationships between the compartments of the cell, it is helpful to consider how they might have evolved. The precursors of the first eucaryotic cells are thought to have been simple organisms that resembled bacteria, which generally have a plasma membrane but no internal membranes. The plasma membrane in such cells therefore provides all membrane-dependent functions, including the pumping of ions, ATP synthesis, protein secretion, and lipid synthesis. Typical present-day eucaryotic cells are 10–30 times larger in linear dimension and 1000–10,000 times greater in volume than a typical bacterium such as E. coli. The profusion of internal membranes can be seen in part as an adaptation to this increase in size: the eucaryotic cell has a much smaller ratio of surface area to volume, and its area of plasma membrane is presumably too small to sustain the many vital functions for which membranes are required. The extensive internal membrane systems of a eucaryotic cell alleviate this imbalance.

The evolution of internal membranes evidently accompanied the specialization of membrane function. Consider, for example, the generation of thylakoid vesicles in chloroplasts. These vesicles form during the development of chloroplasts from proplastids in the green leaves of plants. Proplastids are small precursor organelles that are present in all immature plant cells. They are surrounded by a double membrane and develop according to the needs of the differentiated cells: they develop into chloroplasts in leaf cells, for example, and into organelles that store starch, fat, or pigments in other cell types (Figure 12-3A). In the process of differentiating into chloroplasts, specialized membrane patches form and pinch off from the inner membrane of the proplastid. The vesicles that pinch off form a new specialized compartment, the thylakoid, that harbors all of the chloroplast's photosynthetic machinery (Figure 12-3B).

Other compartments in eucaryotic cells may have originated in a conceptually similar way (Figure 12-4A). Pinching off of specialized intracellular membrane structures from the plasma membrane, for example, would create organelles with an interior that is topologically equivalent to the exterior of the cell. We shall see that this topological relationship holds for all of the organelles involved in the secretory and endocytic pathways, including the ER, Golgi apparatus, endosomes, and lysosomes. We can therefore think of all of these organelles as members of the same family. As we discuss in detail in the next chapter, their interiors communicate extensively with one another and with the outside of the cell via transport vesicles that bud off from one organelle and fuse with another (Figure 12-5).

As described in Chapter 14, mitochondria and plastids differ from the other membrane-enclosed organelles in containing their own genomes. The nature of these genomes, and the close resemblance of the proteins in these organelles to those in some present-day bacteria, strongly suggest that mitochondria and plastids evolved from bacteria that were engulfed by other cells with which they initially lived in symbiosis (discussed in Chapters 1 and 14). According to the hypothetical scheme shown in Figure 12-4B, the inner membrane of mitochondria and plastids corresponds to the original plasma membrane of the bacterium, while the lumen of these organelles evolved from the bacterial cytosol. As might be expected from such an endocytic origin, these two organelles are surrounded by a double membrane, and they remain isolated from the extensive vesicular traffic that connects the interiors of most of the other membrane-enclosed organelles to each other and to the outside of the cell.

The evolutionary scheme described above groups the intracellular compartments in eucaryotic cells into four distinct families: (1) the nucleus and the cytosol, which communicate through nuclear pore complexes and are thus topologically continuous (although functionally distinct); (2) all organelles that function in the secretory and endocytic pathways—including the ER, Golgi apparatus, endosomes, lysosomes, the numerous classes of transport intermediates such as transport vesicles, and possibly peroxisomes; (3) the mitochondria; and (4) the plastids (in plants only).

Proteins Can Move Between Compartments in Different Ways

All proteins begin being synthesized on ribosomes in the cytosol, except for the few that are synthesized on the ribosomes of mitochondria and plastids. Their subsequent fate depends on their amino acid sequence, which can contain sorting signals that direct their delivery to locations outside the cytosol. Most proteins do not have a sorting signal and consequently remain in the cytosol as permanent residents. Many others, however, have specific sorting signals that direct their transport from the cytosol into the nucleus, the ER, mitochondria, plastids, or peroxisomes; sorting signals can also direct the transport of proteins from the ER to other destinations in the cell.

To understand the general principles by which sorting signals operate, it is important to distinguish three fundamentally different ways by which proteins move from one compartment to another. These three mechanisms are described below, and their sites of action in the cell are outlined in Figure 12-6. The first two mechanisms are detailed in this chapter, while the third (green arrows in Figure 12-6) is the subject of Chapter 13.

• 1

  In gated transport, the protein traffic between the cytosol and nucleus occurs between topologically equivalent spaces, which are in continuity through the nuclear pore complexes. The nuclear pore complexes function as selective gates that actively transport specific macromolecules and macromolecular assemblies, although they also allow free diffusion of smaller molecules.

• 2

  In transmembrane transport, membrane-bound protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct. The transported protein molecule usually must unfold to snake through the translocator. The initial transport of selected proteins from the cytosol into the ER lumen or from the cytosol into mitochondria, for example, occurs in this way.

• 3
  In vesicular transport, membrane-enclosed transport intermediates—which may be small, spherical transport vesicles or larger, irregularly shaped organelle fragments—ferry proteins from one compartment to another. The transport vesicles and fragments become loaded with a cargo of molecules derived from the lumen of one compartment as they pinch off from its membrane; they discharge their cargo into a second compartment by fusing with that compartment (Figure 12-7). The transfer of soluble proteins from the ER to the Golgi apparatus, for example, occurs in this way. Because the transported proteins do not cross a membrane, vesicular transport can move proteins only between compartments that are topologically equivalent (see Figure 12-5). We discuss vesicular transport in detail in Chapter 13.

Each of the three modes of protein transfer is usually guided by sorting signals in the transported protein that are recognized by complementary receptor proteins. If a large protein is to be imported into the nucleus, for example, it must possess a sorting signal that is recognized by receptor proteins that guide it through the nuclear pore complex. If a protein is to be transferred directly across a membrane, it must possess a sorting signal that is recognized by the translocator in the membrane to be crossed. Likewise, if a protein is to be loaded into a certain type of vesicle or retained in certain organelles, its sorting signal must be recognized by a complementary receptor in the appropriate membrane.

Signal Sequences and Signal Patches Direct Proteins to the Correct Cellular Address

There are at least two types of sorting signals in proteins. One type resides in a continuous stretch of amino acid sequence, typically 15–60 residues long. Some of these signal sequences are removed from the finished protein by specialized signal peptidases once the sorting process has been completed. The other type consists of a specific three-dimensional arrangement of atoms on the protein's surface that forms when the protein folds up. The amino acid residues that comprise this signal patch can be distant from one another in the linear amino acid sequence, and they generally persist in the finished protein (Figure 12-8). Signal sequences are used to direct proteins from the cytosol into the ER, mitochondria, chloroplasts, and peroxisomes, and they are also used to transport proteins from the nucleus to the cytosol and from the Golgi apparatus to the ER. The sorting signals that direct proteins into the nucleus from the cytosol can be either short signal sequences or longer sequences that are likely to fold into signal patches. Signal patches also direct newly synthesized degradative enzymes into lysosomes.

Each signal sequence specifies a particular destination in the cell. Proteins destined for initial transfer to the ER usually have a signal sequence at their N terminus, which characteristically includes a sequence composed of about 5–10 hydrophobic amino acids. Many of these proteins will in turn pass from the ER to the Golgi apparatus, but those with a specific sequence of four amino acids at their C terminus are recognized as ER residents and are returned to the ER. Proteins destined for mitochondria have signal sequences of yet another type, in which positively charged amino acids alternate with hydrophobic ones. Finally, many proteins destined for peroxisomes have a signal peptide of three characteristic amino acids at their C terminus.

Some specific signal sequences are presented in Table 12-3. The importance of each of these signal sequences for protein targeting has been shown by experiments in which the peptide is transferred from one protein to another by genetic engineering techniques. Placing the N-terminal ER signal sequence at the beginning of a cytosolic protein, for example, redirects the protein to the ER. Signal sequences are therefore both necessary and sufficient for protein targeting. Even though their amino acid sequences can vary greatly, the signal sequences of all proteins having the same destination are functionally interchangeable, and physical properties, such as hydrophobicity, often seem to be more important in the signal-recognition process than the exact amino acid sequence.

Signal patches are far more difficult to analyze than signal sequences, so less is known about their structure. Because they often result from a complex three-dimensional protein-folding pattern, they cannot be easily transferred experimentally from one protein to another.

Both types of sorting signals are recognized by complementary sorting receptors that guide proteins to their appropriate destination, where the receptors unload their cargo. The receptors function catalytically: after completing one round of targeting, they return to their point of origin to be reused. Most sorting receptors recognize classes of proteins rather than just an individual protein species. They therefore can be viewed as public transportation systems dedicated to delivering groups of components to their correct location in the cell.

The main ways of studying how proteins are directed from the cytosol to a specific compartment and how they are translocated across membranes are illustrated in Panel 12-1.

Most Membrane-enclosed Organelles Cannot Be Constructed From Scratch: They Require Information in the Organelle Itself

When a cell reproduces by division, it has to duplicate its membrane-enclosed organelles. In general, cells do this by enlarging the existing organelles by incorporating new molecules into them; the enlarged organelles then divide and are distributed to the two daughter cells. Thus, each daughter cell inherits from its mother a complete set of specialized cell membranes. This inheritance is essential because a cell could not make such membranes from scratch. If the ER were completely removed from a cell, for example, how could the cell reconstruct it? As we shall discuss later, the membrane proteins that define the ER and perform many of its functions are themselves products of the ER. A new ER could not be made without an existing ER or, at the very least, a membrane that specifically contains the protein translocators required to import selected proteins into the ER from the cytosol (including the ER-specific translocators themselves). The same is true for mitochondria, plastids, and peroxisomes (see Figure 12-6).

Thus, it seems that the information required to construct a membrane-enclosed organelle does not reside exclusively in the DNA that specifies the organelle's proteins. Epigenetic information in the form of at least one distinct protein that preexists in the organelle membrane is also required, and this information is passed from parent cell to progeny cell in the form of the organelle itself. Presumably, such information is essential for the propagation of the cell's compartmental organization, just as the information in DNA is essential for the propagation of the cell's nucleotide and amino acid sequences.

As we discuss in more detail in Chapter 13, however, the ER sheds a constant stream of membrane vesicles that incorporate only specific proteins and therefore have a different composition from the ER itself. Similarly, the plasma membrane constantly produces specialized endocytic vesicles. Thus, some membrane-enclosed compartments can form from other organelles and do not have to be inherited at cell division.

Summary

Eucaryotic cells contain intracellular membranes that enclose nearly half the cell's total volume in separate intracellular compartments called organelles. The main types of membrane-enclosed organelles present in all eucaryotic cells are the endoplasmic reticulum, Golgi apparatus, nucleus, mitochondria, lysosomes, endosomes, and peroxisomes; plant cells also contain plastids, such as chloroplasts. Each organelle contains a distinct set of proteins that mediate its unique functions.

Each newly synthesized organelle protein must find its way from a ribosome in the cytosol, where it is made, to the organelle where it functions. It does so by following a specific pathway, guided by signals in its amino acid sequence that function as signal sequences or signal patches. Signal sequences and patches are recognized by complementary sorting receptors that deliver the protein to the appropriate target organelle. Proteins that function in the cytosol do not contain sorting signals and therefore remain there after they are synthesized.

During cell division, organelles such as the ER and mitochondria are distributed intact to each daughter cell. These organelles contain information that is required for their construction so that they cannot be made from scratch.

Nuclear Pore Complexes Perforate the Nuclear Envelope

The nuclear envelope of all eucaryotes is perforated by large, elaborate structures known as nuclear pore complexes. In animal cells, each complex has an estimated molecular mass of about 125 million and is thought to be composed of more than 50 different proteins, called nucleoporins, that are arranged with a striking octagonal symmetry (Figure 12-10).

In general, the more active the nucleus is in transcription, the greater the number of pore complexes its envelope contains. The nuclear envelope of a typical mammalian cell contains 3000–4000 pore complexes. If the cell is synthesizing DNA, it needs to import about 10⁶ histone molecules from the cytosol every 3 minutes to package the newly made DNA into chromatin, which means that, on average, each pore complex needs to transport about 100 histone molecules per minute. If the cell is growing rapidly, each complex also needs to transport about 6 newly assembled large and small ribosomal subunits per minute from the nucleus, where they are produced, to the cytosol, where they are used. And that is only a very small part of the total traffic that passes through the pore complexes.

Each pore complex contains one or more open aqueous channels through which small water-soluble molecules can passively diffuse. The effective size of these channels has been determined by injecting labeled water-soluble molecules of different sizes into the cytosol and then measuring their rate of diffusion into the nucleus. Small molecules (5000 daltons or less) diffuse in so fast that the nuclear envelope can be considered to be freely permeable to them. A protein of 17,000 daltons takes 2 minutes to equilibrate between the cytosol and the nucleus, whereas proteins larger than 60,000 daltons are hardly able to enter the nucleus at all. A quantitative analysis of such data suggests that the nuclear pore complex contains a pathway for free diffusion equivalent to a water-filled cylindrical channel about 9 nm in diameter and 15 nm long; such a channel would occupy only a small fraction of the total volume of the pore complex (Figure 12-11).

Because many cell proteins are too large to pass by diffusion through the nuclear pore complexes, the nuclear envelope enables the nuclear compartment and the cytosol to maintain different complements of proteins. Mature cytosolic ribosomes, for example, are about 30 nm in diameter and thus cannot diffuse through the 9 nm channels; their exclusion from the nucleus ensures that protein synthesis is confined to the cytosol. But how does the nucleus export newly made ribosomal subunits or import large molecules, such as DNA and RNA polymerases, which have subunit molecular weights of 100,000–200,000 daltons? As we discuss next, these and many other protein and RNA molecules bind to specific receptor proteins that ferry them actively through nuclear pore complexes.

Nuclear Localization Signals Direct Nuclear Proteins to the Nucleus

When proteins are experimentally extracted from the nucleus and reintroduced into the cytosol (e.g., through experimentally induced perforations in the plasma membrane), even the very large ones reaccumulate efficiently in the nucleus. The selectivity of this nuclear import process resides in nuclear localization signals (NLSs), which are present only in nuclear proteins. The signals have been precisely defined in numerous nuclear proteins by using recombinant DNA technology (Figure 12-12). As mentioned earlier, they can be either signal sequences or signal patches. In many nuclear proteins they consist of one or two short sequences that are rich in the positively charged amino acids lysine and arginine (see Table 12-3, p. 667), the precise sequence varying for different nuclear proteins. Other nuclear proteins contain different signals, some of which are not yet characterized.

The signals characterized this far can be located almost anywhere in the amino acid sequence and are thought to form loops or patches on the protein surface. Many function even when linked as short peptides to lysine side chains on the surface of a cytosolic protein, suggesting that the precise location of the signal within the amino acid sequence of a nuclear protein is not important.

The transport of nuclear proteins through nuclear pore complexes can be directly visualized by coating gold particles with a nuclear localization signal, injecting the particles into the cytosol, and then following their fate by electron microscopy (Figure 12-13). Studies with various sizes of gold beads indicate that the opening can dilate up to about 26 nm in diameter during the transport process. A structure in the center of the nuclear pore complex seems to function like a close-fitting diaphragm that opens just the right amount to let transport substrates pass (see Figure 12-11). The molecular basis of the gating mechanism remains a mystery.

The mechanism of macromolecular transport across nuclear pore complexes is fundamentally different from the transport mechanisms involved in protein transfer across the membranes of other organelles, because it occurs through a large aqueous pore rather than through a protein transporter spanning one or more lipid bilayers. For this reason, nuclear proteins can be transported through a pore complex while they are in a fully folded conformation. Likewise, a newly formed ribosomal subunit is transported out of the nucleus as an assembled particle. By contrast, proteins have to be extensively unfolded during their transport into most other organelles, as we discuss later. In the electron microscope, however, very large particles traversing the pore seem to become constricted as they squeeze through the nuclear pore complex, indicating that at least some of them must undergo restructuring during transport. This has been most extensively studied for the export of some very large mRNAs, as discussed in Chapter 6 (see Figure 6-39).

Nuclear Import Receptors Bind Nuclear Localization Signals and Nucleoporins

To initiate nuclear import, most nuclear localization signals must be recognized by nuclear import receptors, which are encoded by a family of related genes. Each family member encodes a receptor protein that is specialized for the transport of a group of nuclear proteins sharing structurally similar nuclear localization signals (Figure 12-14A).

The import receptors are soluble cytosolic proteins that bind both to the nuclear localization signal on the protein to be transported and to nucleoporins, some of which form the tentaclelike fibrils that extend into the cytosol from the rim of the nuclear pore complexes. The fibrils and many other nucleoporins contain a large number of short amino-acid repeats that contain phenylalanine and glycine and are therefore called FG-repeats (named after the one-letter code for amino acids, discussed in Chapter 5). FG-repeats serve as binding sites for the import receptors. They are thought to line the path through the nuclear pore complexes taken by the import receptors and their bound cargo proteins. These protein complexes move along the path by repeatedly binding, dissociating, and then re-binding to adjacent repeat sequences. Once in the nucleus, the import receptors dissociate from their cargo and are returned to the cytosol.

Nuclear import receptors do not always bind to nuclear proteins directly. Additional adaptor proteins are sometimes used that bridge between the import receptors and the nuclear localization signals on the proteins to be transported. Surprisingly, the adaptor proteins are structurally related to nuclear import receptors, suggesting a common evolutionary origin. The combined use of import receptors and adaptors allows a cell to recognize the broad repertoire of nuclear localization signals that are displayed on nuclear proteins.

Nuclear Export Works Like Nuclear Import, But in Reverse

The nuclear export of large molecules, such as new ribosomal subunits and RNA molecules, also occurs through nuclear pore complexes and depends on a selective transport system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors. These receptors bind both the export signal and nucleoporins to guide their cargo through the pore complex to the cytosol.

Nuclear export receptors are structurally related to nuclear import receptors, and they are encoded by the same gene family of nuclear transport receptors, or karyopherins. In yeast, there are 14 genes encoding members of this family; in animal cells the number is significantly larger. From their amino acid sequence alone, it is often not possible to distinguish whether a particular family member works as a nuclear import or nuclear export receptor. It comes as no surprise, therefore, that the import and export transport systems work in similar ways but in opposite directions: the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse, while the export receptors function in reverse.

If gold spheres similar to those used in the experiments shown in Figure 12-13 are coated with small RNA molecules (tRNA or ribosomal 5S RNA) and injected into the nucleus of a cultured cell, they are rapidly transported through the nuclear pore complexes into the cytosol. Using two sizes of gold particles, one coated with RNA and injected into the nucleus and the other coated with nuclear localization signals and injected into the cytosol, it can be shown that a single pore complex conducts traffic in both directions. How a pore complex coordinates the bidirectional flow of macromolecules to avoid congestion and head-on collisions is not known.

The Ran GTPase Drives Directional Transport Through Nuclear Pore Complexes

The import of nuclear proteins through the pore complex concentrates specific proteins in the nucleus, thereby increasing order in the cell, which must consume energy (discussed in Chapter 2). The energy is thought to be provided by the hydrolysis of GTP by the monomeric GTPase Ran. Ran is found in both the cytosol and the nucleus, and it is required for both the nuclear import and export systems.

Like other GTPases, Ran is a molecular switch that can exist in two conformational states, depending on whether GDP or GTP is bound (discussed in Chapter 3). Conversion between the two states is triggered by two Ran-specific regulatory proteins: a cytosolic GTPase-activating protein (GAP) that triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) that promotes the exchange of GDP for GTP and thus converts Ran-GDP to Ran-GTP. Because Ran-GAP is located in the cytosol and Ran-GEF is located in the nucleus, the cytosol primarily contains Ran-GDP, and the nucleus primarily contains Ran-GTP (Figure 12-15).

This gradient of the two conformational forms of Ran drives nuclear transport in the appropriate direction (Figure 12-16). Docking of nuclear import receptors to FG-repeats on the cytosolic side of the nuclear pore complex, for example, occurs only when these receptors are loaded with an appropriate cargo. The import receptors with their bound cargo then move along tracks lined by FG-repeat sequences until they reach the nuclear side of the pore complex, where Ran-GTP binding causes the import receptors to release their cargo (Figure 12-17). By favoring cargo-dependent loading of import receptors onto the FG-repeat track in the cytosol and Ran-GTP-dependent cargo release in the nucleus, the nuclear localization of Ran-GTP imposes directionality.

Having discharged its cargo in the nucleus, the empty import receptor with Ran-GTP bound is transported back through the pore complex to the cytosol. There, two cytosolic proteins, Ran Binding Protein and Ran-GAP collaborate to convert Ran-GTP to Ran-GDP. The Ran Binding Protein first displaces Ran-GTP from the import receptor, which allows Ran-GAP to trigger Ran to hydrolyze its bound GTP. The Ran-GDP then dissociates from the Ran Binding Protein and is reimported into the nucleus, thereby completing the cycle.

Nuclear export occurs by a similar mechanism, except that Ran-GTP in the nucleus promotes cargo binding to the export receptor and the binding of the loaded receptor to the nuclear side of the pore complex. Once in the cytosol, Ran encounters Ran-GAP and Ran Binding Protein and hydrolyses its bound GTP. The export receptor then releases both its cargo and Ran-GDP in the cytosol and dissociates from the pore complex, and free export receptors are returned to the nucleus to complete the cycle (see Figure 12-16).

Transport Between the Nucleus and Cytosol Can Be Regulated by Controlling Access to the Transport Machinery

Some proteins, such as those that bind newly made mRNAs in the nucleus, contain both nuclear localization and nuclear export signals. These proteins continually shuttle between the nucleus and the cytosol. The steady-state localization of such shuttling proteins is determined by the relative rates of their import and export. If the rate of import exceeds the rate of export, a protein will be located primarily in the nucleus. Conversely, if the rate of export exceeds the rate of import, a protein will be located primarily in the cytosol. Thus, changing the rate of import, export, or both, can change the location of a protein.

Some shuttling proteins move continuously in and out of the nucleus. In other cases, however, the transport is stringently controlled. As discussed in Chapter 7, the activity of some gene regulatory proteins is controlled by keeping them out of the nuclear compartment until they are needed there (Figure 12-18). In many cases, this control depends on the regulation of nuclear localization and export signals; these can be turned on or off, often by phosphorylation of adjacent amino acids (Figure 12-19).

Other gene regulatory proteins are bound to inhibitory cytosolic proteins that either anchor them in the cytosol (through interactions with the cytoskeleton or with specific organelles), or mask their nuclear localization signals so that they are unable to interact with nuclear import receptors. When the cell receives an appropriate stimulus, the gene regulatory protein is released from its cytosolic anchor or mask and is transported into the nucleus. One important example is the latent gene regulatory protein that controls the expression of proteins involved in cholesterol metabolism. The protein is made and stored in an inactive form as a transmembrane protein in the ER. When deprived of cholesterol, the cell activates specific proteases that cleave the protein, releasing its cytosolic domain. This domain is then imported into the nucleus, where it activates the transcription of genes required for cholesterol import and synthesis.

Cells control the export of RNA from the nucleus in a similar way. Messenger RNAs become bound to proteins that are loaded onto the RNA as transcription and splicing proceed. These proteins contain nuclear export signals that are recognized by export receptors that guide the RNA out of the nucleus through nuclear pore complexes. Upon entry into the cytosol, the proteins coating the RNA are stripped off and rapidly returned to the nucleus. Other RNAs, such as snRNAs and tRNAs, are exported by different sets of nuclear export receptors.

Incompletely processed pre-mRNAs are actively retained in the nucleus, anchored to the nuclear transcription and splicing machinery, which releases an RNA molecule only after its processing is completed. Genetic studies in yeast show that a mutant pre-mRNA that cannot properly engage with the splicing machinery is improperly exported as an unspliced molecule.

The Nuclear Envelope Is Disassembled During Mitosis

The nuclear lamina is a meshwork of interconnected protein subunits called nuclear lamins. The lamins are a special class of intermediate filament proteins (discussed in Chapter 16) that polymerize into a two-dimensional lattice (Figure 12-20). The nuclear lamina gives shape and stability to the nuclear envelope, to which it is anchored by attachment to both the nuclear pore complexes and integral membrane proteins of the inner nuclear membrane. The lamina also interacts directly with chromatin, which itself interacts with the integral membrane proteins of the inner nuclear membrane. Together with the lamina, these membrane proteins provide structural links between the DNA and the nuclear envelope.

When a nucleus disassembles during mitosis, the nuclear lamina depolymerizes. The disassembly is at least partly a consequence of direct phosphorylation of the nuclear lamins by the cyclin-dependent kinase activated at the onset of mitosis (discussed in Chapter 17). At the same time, proteins of the inner nuclear membrane are phosphorylated, and the nuclear pore complexes disassemble and disperse in the cytosol. Nuclear envelope membrane proteins—no longer tethered to the pore complexes, lamina, or chromatin—diffuse throughout the ER membrane. Together, these events break down the barriers that normally separate the nucleus and cytosol, and these nuclear proteins that are not bound to membranes or chromosomes intermix completely with the cytosol of the dividing cell (Figure 12-21).

Later in mitosis (in late anaphase), the nuclear envelope reassembles on the surface of the chromosomes, as inner nuclear membrane proteins and dephosphorylated lamins rebind to chromatin. ER membranes wrap around groups of chromosomes and continue fusing until a sealed nuclear envelope is reformed. During this process, the nuclear pore complexes also reassemble and start actively reimporting proteins that contain nuclear localization signals. Because the nuclear envelope is initially closely applied to the surface of the chromosomes, the newly formed nucleus excludes all proteins except those initially bound to the mitotic chromosomes and those that are selectively imported through nuclear pore complexes. In this way, all other large proteins are kept out of the newly assembled nucleus.

Nuclear localization signals are not cleaved off after transport into the nucleus. This is presumably because nuclear proteins need to be imported repeatedly, once after every cell division. In contrast, once a protein molecule has been imported into any of the other membrane-enclosed organelles, it is passed on from generation to generation within that compartment and need never be translocated again; the signal sequence on these molecules is often removed after protein translocation.

Summary

The nuclear envelope consists of an inner and an outer nuclear membrane. The outer membrane is continuous with the ER membrane, and the space between it and the inner membrane is continuous with the ER lumen. RNA molecules, which are made in the nucleus, and ribosomal subunits, which are assembled there, are exported to the cytosol, while all the proteins that function in the nucleus are synthesized in the cytosol and are then imported. The extensive traffic of materials between the nucleus and cytosol occurs through nuclear pore complexes, which provide a direct passageway across the nuclear envelope.

Proteins containing nuclear localization signals are actively transported inward through the nuclear pore complexes, while RNA molecules and newly made ribosomal subunits contain nuclear export signals that direct their active transport outward through the pore complexes. Some proteins, including nuclear import and export receptors, continually shuttle between the cytosol and nucleus. The GTPase Ran, provides directionality for nuclear transport. The transport of nuclear proteins and RNA molecules through the pore complexes can be regulated by denying these molecules access to the transport machinery. Because nuclear localization signals are not removed, nuclear proteins can be imported repeatedly, as is required each time that the nucleus reassembles after mitosis.

Translocation into the Mitochondrial Matrix Depends on a Signal Sequence and Protein Translocators

Proteins imported into the matrix of mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes. Thus, in contrast to the protein translocation into the ER described later, mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria by a posttranslational mechanism. Most of the mitochondrial precursor proteins have a signal sequence at their N terminus that is rapidly removed after import by a protease (the signal peptidase) in the mitochondrial matrix. The signal sequences are both necessary and sufficient for import of the proteins that contain them: through the use of genetic engineering techniques, these signals can be linked to any cytosolic protein to direct the protein into the mitochondrial matrix. Sequence comparisons and physical studies of different matrix signal sequences suggest that their common feature is the propensity to fold into an amphipathic α helix, in which positively charged residues are clustered on one side of the helix, while uncharged hydrophobic residues are clustered on the opposite side (Figure 12-23). This configuration—rather than a precise amino acid sequence—is recognized by specific receptor proteins that initiate protein translocation.

Protein translocation across mitochondrial membranes is mediated by multi-subunit protein complexes that function as protein translocators: the TOM complex functions across the outer membrane, and two TIM complexes, the TIM23 and TIM22 complexes, function across the inner membrane (Figure 12-24). TOM and TIM stand for translocase of the outer and inner mitochondrial membranes, respectively. These complexes contain some components that act as receptors for mitochondrial precursor proteins and other components that form the translocation channel. The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins. It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane. The TIM23 complex then transports some of these proteins into the matrix space, while helping to insert transmembrane proteins into the inner membrane. The TIM22 complex mediates the insertion of a subclass of inner membrane proteins, including the carrier protein that transports ADP, ATP, and phosphate. A third protein translocator in the inner mitochondrial membrane, the OXA complex, mediates the insertion of inner membrane proteins that are synthesized within the mitochondria. It also helps to insert some proteins that are initially transported into the matrix by the TOM and TIM complexes.

Mitochondrial Precursor Proteins Are Imported as Unfolded Polypeptide Chains

Almost everything we know about the molecular mechanism of protein import into mitochondria has been learned from analyses of cell-free, reconstituted transport systems. Mitochondria are first purified by differential centrifugation of homogenized cells and are then incubated with radiolabeled mitochondrial precursor proteins, which are generally taken up rapidly and efficiently. By changing the incubation conditions, it is possible to establish the biochemical requirements for the transport.

Mitochondrial precursor proteins do not fold into their native structures after they are synthesized; instead, they remain unfolded through interactions with other proteins in the cytosol. Some of these interacting proteins are general chaperone proteins belonging to the hsp70 family (discussed in Chapter 6), whereas others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences. All these interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex in the outer mitochondrial membrane. As a first step in the import process, the mitochondrial precursor proteins bind to import receptor proteins of the TOM complex, which recognize the mitochondrial signal sequences. The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the translocation channel.

Mitochondrial Precursor Proteins Are Imported into the Matrix at Contact Sites That Join the Inner and Outer Membranes

In principle, a protein could reach the mitochondrial matrix by crossing the two membranes one at a time, or it could pass through both at once. To distinguish between these possibilities, a cell-free mitochondrial import system was cooled to a low temperature, arresting the proteins at an intermediate step in the translocation process. The proteins that accumulated at this step already had their N-terminal signal sequence removed by the matrix signal peptidase, indicating that their N terminus must be in the matrix space. Yet, the bulk of the protein could still be attacked from outside the mitochondria by externally added proteolytic enzymes (Figure 12-25). This result demonstrates that the precursor proteins can pass through both mitochondrial membranes at once to enter the matrix (Figure 12-26). It is thought that the TOM complex first transports the mitochondrial targeting signal across the outer membrane. Once it reaches in the intermembrane space, the targeting signal binds to a TIM complex, opening the channel in the complex through which the polypeptide chain either enters the matrix or inserts into the inner membrane. Electron microscopists have noted numerous contact sites at which the inner and outer mitochondrial membranes are closely apposed, and it seems likely that translocation occurs at or near these sites.

Although the functions of the TOM and TIM complexes are usually coupled to allow protein transport across both membranes at the same time, both protein types of translocator can work independently. The TOM complex in isolated outer membranes, for example, can translocate the signal sequence of precursor proteins across the membrane. Similarly, mitochondria with experimentally disrupted outer membranes, and therefore with the TIM23 complex exposed on their surface, efficiently import precursor proteins into the matrix space. Despite the independent functional roles of the TOM and TIM translocators, the two mitochondrial membranes at contact sites may be permanently held together by the TIM23 complex, which spans both membranes (see Figure 12-24).

ATP Hydrolysis and a H⁺ Gradient are Used to Drive Protein Import into Mitochondria

Directional transport requires energy. In most biological systems, energy is supplied by ATP hydrolysis. Mitochondrial protein import is fueled by ATP hydrolysis at two discrete sites, one outside the mitochondria and one in the matrix (Figure 12-27). In addition, another energy source is required: an electrochemical H⁺ gradient across the inner mitochondrial membrane.

The first requirement for energy occurs at the initial stage of the translocation process, when the unfolded precursor protein, associated with chaperone proteins, interacts with the mitochondrial import receptors. As discussed in Chapter 6, the release of newly synthesized polypeptides from the hsp70 family of chaperone proteins requires ATP hydrolysis. Experimentally, the requirement for hsp70 and ATP in the cytosol can be bypassed if the precursor protein is artificially unfolded prior to adding it to purified mitochondria.

Once the signal sequence has passed through the TOM complex and has become bound to either TIM complex, further translocation through the TIM requires an electrochemical H⁺ gradient across the inner membrane. The electrochemical gradient is maintained by the pumping of H⁺ from the matrix to the intermembrane space, driven by electron transport processes in the inner membrane. By contrast, the outer mitochondrial membrane, like that of Gram-negative bacteria (see Figure 11-17), contains a pore-forming protein called porin and is thus freely permeable to inorganic ions and metabolites (but not to most proteins), so that ion gradients cannot be maintained across it. The energy in the electrochemical H⁺ gradient across the inner membrane is not only used to help drive most of the cell's ATP synthesis; it is also used to drive the translocation of the targeting signals through the TIM complexes. The precise mechanism by which this occurs is not known, but it is possible that the electrical components of the gradient (the membrane potential, see Figure 14-13) helps to drive the positively charged signal sequence into the matrix by electrophoresis.

Hsp70 chaperone proteins in the matrix space also have a role in the translocation process, and they are the third point in the import process at which ATP is consumed, as we discuss next.

Repeated Cycles of ATP Hydrolysis by Mitochondrial Hsp70 Complete the Import Process

We know that mitochondrial hsp70 is crucial to the import process, because mitochondria containing mutant forms of the protein fail to import precursor proteins. Like its cytosolic cousin, mitochondrial hsp70 has a high affinity for unfolded polypeptide chains and it binds tightly to an imported protein as soon as it emerges from the translocator in the matrix. The hsp70 then releases the protein in an ATP-dependent step. This energy-driven cycle of binding and subsequent release is thought to provide the final driving force needed to complete protein import after a protein has initially inserted into the TIM23 complex (see Figure 12-27).

Two models have been proposed to explain how ATP hydrolysis by mitochondrial hsp70 drives protein import. In both models, hsp70 proteins are closely associated with the TIM23 complex, which deposits them onto the translocating polypeptide chain as it emerges into the matrix. In the thermal ratchet model (Figure 12-28A), the emerging chain slides back and forth in the TIM23 translocation channel by thermal motion. Each time a sufficiently long portion of the chain is exposed in the matrix, an hsp70 molecule binds to it, preventing further backsliding and thereby making the movement directional. Thus, a hand-over-hand binding of multiple hsp70 proteins translocates the polypeptide chain into the matrix. In the cross-bridge ratchet model (Figure 12-28B), the hsp70 proteins that bind to the emerging polypeptide chain undergo a conformational change, driven by ATP hydrolysis, that actively pulls a segment of the polypeptide chain into the matrix. A new hsp70 molecule can then bind to the segment just pulled in and repeat the cycle. In both models, therefore, hsp70 functions as a ratchet that prevents backsliding of the emerging polypeptide chain.

After the initial interaction with mitochondrial hsp70, many imported proteins are passed on to another chaperone protein, mitochondrial hsp60. As discussed in Chapter 6, hsp60 provides a chamber for the unfolded polypeptide chain that facilitates its folding by binding and releasing it through cycles of ATP hydrolysis.

Protein Transport into the Inner Mitochondrial Membrane and the Intermembrane Space Requires Two Signal Sequences

Proteins that are integrated into the inner mitochondrial membrane or that operate in the intermembrane space are initially transported from the cytosol by the same mechanism that transports proteins into the matrix. In some cases they are first transferred into the matrix (see Figure 12-26). A hydrophobic amino acid sequence, however, is strategically placed after the N-terminal signal sequence that guides import into the matrix. Once the N-terminal signal sequence has been removed by the matrix signal peptidase, the hydrophobic sequence functions as a new N-terminal signal sequence to translocate the protein from the matrix into or across the inner membrane, using the OXA complex as the translocator (see Figure 12-24). The OXA complex is also used to insert proteins encoded in the mitochondrion into the inner membrane (Figure 12-29A). Closely related translocators are found in the plasma membranes of bacteria and in the thylakoids of chloroplasts, where they are thought to help to insert membrane proteins by a similar mechanism.

An alternative route to the inner membrane avoids excursion into the matrix altogether (Figure 12-29B). In this case, the TIM23 translocator in the inner membrane binds to the hydrophobic sequence that follows the N-terminal signal sequence and initiates import, causing it to act as a stop-transfer sequence that prevents further translocation across the inner membrane. After the N-terminal signal sequence has been cleaved off, the remainder of the protein is pulled through the TOM complex in the outer membrane into the intermembrane space. Different proteins use one or the other of these two pathways to the inner membrane or intermembrane space.

Proteins destined for the intermembrane space are first inserted via their hydrophobic signal sequence into the inner membrane, and then cleaved by a signal peptidase in the intermembrane space to release the mature polypeptide chain as a soluble protein (Figure 12-29C). Many of these proteins attach as peripheral membrane proteins to the outer surface of the inner membrane, where they form subunits of protein complexes that also contain transmembrane proteins.

Mitochondria are the principal site of ATP synthesis in the cell, but they also contain many metabolic enzymes, such as those of the citric acid cycle. Thus, in addition to proteins, mitochondria must also transport small metabolites across their membranes. While the outer membrane contains porins that make the membrane freely permeable to small molecules, the inner membrane does not. Instead, the transport of a vast number of small molecules across the inner membrane is mediated by a family of metabolite-specific carrier proteins. In yeast cells, these proteins comprise a family of 35 different proteins, of which the most abundant are those that transport ADP and ATP, or phosphate. These carrier proteins in the inner membrane are multipass transmembrane proteins, which do not have cleavable signal sequences at their N termini but, instead, contain internal signal sequences. These proteins cross the TOM complex in the outer membrane and are inserted into the inner membrane by the TIM22 complex (Figure 12-29D). Their integration into the inner membrane requires the electrochemical H⁺ gradient, but not mitochondrial hsp70 or ATP. The energetically favorable partitioning of the hydrophobic transmembrane regions into the inner membrane is likely to help drive integration.

Two Signal Sequences Are Required to Direct Proteins to the Thylakoid Membrane in Chloroplasts

Protein transport into chloroplasts resembles transport into mitochondria in many respects. Both processes occur posttranslationally, use separate translocation complexes in each membrane, occur at contact sites, require energy, and use amphipathic N-terminal signal sequences that are removed after use. With the exception of some of the chaperone molecules, however, the protein components that form the translocation complexes are different. Moreover, whereas mitochondria harness the electrochemical H⁺ gradient across their inner membrane to drive transport, chloroplasts, which have an electrochemical H⁺ gradient across their thylakoid membrane but not their inner membrane, use the hydrolysis of GTP and ATP to power import across their double membrane. The functional similarities may thus result from convergent evolution, reflecting the common requirements for translocation across a double-membrane system.

Although the signal sequences for import into chloroplasts superficially resemble those for import into mitochondria, mitochondria and chloroplasts are both present in the same plant cells, so proteins must choose appropriately between them. In plants, for example, a bacterial enzyme can be directed specifically to mitochondria if it is experimentally joined to an N-terminal signal sequence of a mitochondrial protein; the same enzyme joined to an N-terminal signal sequence of a chloroplast protein ends up in chloroplasts. The different signal sequences can therefore be distinguished by the import receptors on each organelle.

Chloroplasts have an extra membrane-enclosed compartment, the thylakoid. Many chloroplast proteins, including the protein subunits of the photosynthetic system and of the ATP synthase (discussed in Chapter 14), are embedded in the thylakoid membrane. Like the precursors of some mitochondrial proteins, these proteins are transported from the cytosol to their final destination in two steps. First, they pass across the double membrane at contact sites into the matrix space of the chloroplast, called the stroma, and then they are translocated into the thylakoid membrane (or across this membrane into the thylakoid space) (Figure 12-30A). The precursors of these proteins have a hydrophobic thylakoid signal sequence following the N-terminal chloroplast signal sequence. After the N-terminal signal sequence has been used to import the protein into the stroma, it is removed by a stromal signal peptidase (analogous to the matrix signal peptidase in mitochondria). This cleavage unmasks the thylakoid signal sequence, which then initiates transport across the thylakoid membrane. There are at least four routes for proteins to cross or become integrated into the thylakoid membrane, distinguished by their need for different stromal chaperones and energy sources (Figure 12-30B).

Summary

Although mitochondria and chloroplasts have their own genetic systems, they produce only a small proportion of their own proteins. Instead, the two organelles import most of their proteins from the cytosol, using similar mechanisms. In both cases, proteins are imported in an unfolded state. Proteins are translocated into the mitochondrial matrix space by passing through the TOM and TIM complexes at sites of adhesion between the outer and inner membranes known as contact sites. Translocation into mitochondria is driven by both ATP hydrolysis and an electrochemical H⁺gradient across the inner membrane, whereas translocation into chloroplasts is driven solely by the hydrolysis of GTP and ATP.

Chaperone proteins of the cytosolic hsp70 family maintain the precursor proteins in an unfolded, translocation-competent state. A second set of hsp70 proteins in the matrix or stroma bind to the incoming polypeptide chain to pull it into the organelle. Only proteins that contain a specific signal sequence are translocated into mitochondria or chloroplasts. The signal sequence is usually located at the N terminus and is cleaved off after import. Some imported proteins also contain an internal signal sequence that guides their further transport. Transport across or into the inner membrane can occur as a second step if a hydrophobic signal sequence is unmasked when the first signal sequence is removed. In chloroplasts, import from the stroma into the thylakoid likewise requires a second signal sequence and can occur by one of several routes.

Peroxisomes Use Molecular Oxygen and Hydrogen Peroxide to Perform Oxidative Reactions

Peroxisomes are so named because they usually contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from specific organic substrates (designated here as R) in an oxidative reaction that produces hydrogen peroxide (H₂O₂):

Catalase utilizes the H₂O₂ generated by other enzymes in the organelle to oxidize a variety of other substrates—including phenols, formic acid, formaldehyde, and alcohol—by the “peroxidative” reaction: H₂O₂ + R′ H₂ → R′ + 2H₂O. This type of oxidative reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various toxic molecules that enter the bloodstream. About 25% of the ethanol we drink is oxidized to acetaldehyde in this way. In addition, when excess H₂O₂ accumulates in the cell, catalase converts it to H₂O through the reaction:

A major function of the oxidative reactions performed in peroxisomes is the breakdown of fatty acid molecules. In a process called β oxidation, the alkyl chains of fatty acids are shortened sequentially by blocks of two carbon atoms at a time, thereby converting the fatty acids to acetyl CoA. The acetyl CoA is then exported from the peroxisomes to the cytosol for reuse in biosynthetic reactions. In mammalian cells, β oxidation occurs in both mitochondria and peroxisomes; in yeast and plant cells, however, this essential reaction occurs exclusively in peroxisomes.

An essential biosynthetic function of animal peroxisomes is to catalyze the first reactions in the formation of plasmalogens, which are the most abundant class of phospholipids in myelin (Figure 12-32). Deficiency of plasmalogens causes profound abnormalities in the myelination of nerve cells, which is one reason why many peroxisomal disorders lead to neurological disease.

Peroxisomes are unusually diverse organelles, and even in the various cell types of a single organism they may contain different sets of enzymes. They can also adapt remarkably to changing conditions. Yeast cells grown on sugar, for example, have small peroxisomes. But when some yeasts are grown on methanol, they develop large peroxisomes that oxidize methanol; and when grown on fatty acids, they develop large peroxisomes that break down fatty acids to acetyl CoA by β oxidation.

Peroxisomes are also important in plants. Two different types have been studied extensively. One type is present in leaves, where it catalyzes the oxidation of a side product of the crucial reaction that fixes CO₂ in carbohydrate (Figure 12-33A). As discussed in Chapter 14, this process is called photorespiration because it uses up O₂ and liberates CO₂. The other type of peroxisome is present in germinating seeds, where it has an essential role in converting the fatty acids stored in seed lipids into the sugars needed for the growth of the young plant. Because this conversion of fats to sugars is accomplished by a series of reactions known as the glyoxylate cycle, these peroxisomes are also called glyoxysomes (Figure 12-33B). In the glyoxylate cycle, two molecules of acetyl CoA produced by fatty acid breakdown in the peroxisome are used to make succinic acid, which then leaves the peroxisome and is converted into glucose. The glyoxylate cycle does not occur in animal cells, and animals are therefore unable to convert the fatty acids in fats into carbohydrates.

A Short Signal Sequence Directs the Import of Proteins into Peroxisomes

A specific sequence of three amino acids located at the C terminus of many peroxisomal proteins functions as an import signal (see Table 12-3). Other peroxisomal proteins contain a signal sequence near the N terminus. If either of these sequences is experimentally attached to a cytosolic protein, the protein is imported into peroxisomes. The import process is still poorly understood, although it is known to involve soluble receptor proteins in the cytosol that recognize the targeting signals, as well as docking proteins on the cytosolic surface of the peroxisome. At least 23 distinct proteins, called peroxins, participate as components in the process, which is driven by ATP hydrolysis. Oligomeric proteins do not have to unfold to be imported into peroxisomes, indicating that the mechanism is distinct from that used by mitochondria and chloroplasts and at least one soluble import receptor, the peroxin Pex5, accompanies its cargo all the way into peroxisomes and, after cargo release, cycles back out into the cytosol. These aspects of peroxisomal protein import resemble protein tranport into the nucleus.

The importance of this import process and of peroxisomes is demonstrated by the inherited human disease Zellweger syndrome, in which a defect in importing proteins into peroxisomes leads to a severe peroxisomal deficiency. These individuals, whose cells contain “empty” peroxisomes, have severe abnormalities in their brain, liver, and kidneys, and they die soon after birth. One form of this disease has been shown to be due to a mutation in the gene encoding a peroxisomal integral membrane protein, the peroxin Pex2, involved in protein import. A milder inherited peroxisomal disease is caused by a defective receptor for the N-terminal import signal.

Most peroxisomal membrane proteins are made in the cytosol and then insert into the membrane of preexisting peroxisomes. Thus, new peroxisomes are thought to arise from preexisting ones, by organelle growth and fission—as mentioned earlier for mitochondria and plastids, and as we describe below for the ER (Figure 12-34).

Summary

Peroxisomes are specialized for carrying out oxidative reactions using molecular oxygen. They generate hydrogen peroxide, which they use for oxidative purposes—destroying the excess by means of the catalase they contain. Peroxisomes also have an important role in the synthesis of specialized phospholipids required for nerve cell myelination. Like mitochondria and plastids, peroxisomes are thought to be self-replicating organelles. Because they contain no DNA or ribosomes, however, they have to import their proteins from the cytosol. A specific sequence of three amino acids near the C terminus of many of these proteins functions as a peroxisomal import signal. The mechanism of protein import is distinct from that of mitochondria and chloroplasts, and oligomeric proteins can be transported into peroxisomes without unfolding.

Membrane-bound Ribosomes Define the Rough ER

The ER captures selected proteins from the cytosol as they are being synthesized. These proteins are of two types: transmembrane proteins, which are only partly translocated across the ER membrane and become embedded in it, and water-soluble proteins, which are fully translocated across the ER membrane and are released into the ER lumen. Some of the transmembrane proteins function in the ER, but many are destined to reside in the plasma membrane or the membrane of another organelle. The water-soluble proteins are destined either for the lumen of an organelle or for secretion. All of these proteins, regardless of their subsequent fate, are directed to the ER membrane by the same kind of signal sequence and are translocated across it by similar mechanisms.

In mammalian cells, the import of proteins into the ER begins before the polypeptide chain is completely synthesized—that is, import is a co-translational process. This distinguishes the process from the import of proteins into mitochondria, chloroplasts, nuclei, and peroxisomes, which are posttranslational processes. Since one end of the protein is usually translocated into the ER as the rest of the polypeptide chain is being made, the protein is never released into the cytosol and therefore is never in danger of folding up before reaching the translocator in the ER membrane. Thus, in contrast to the posttranslational import of proteins into mitochondria and chloroplasts, chaperone proteins are not required to keep the protein unfolded. The ribosome that is synthesizing the protein is directly attached to the ER membrane. These membrane-bound ribosomes coat the surface of the ER, creating regions termed rough endoplasmic reticulum, or rough ER (Figure 12-36A).

There are therefore two spatially separate populations of ribosomes in the cytosol. Membrane-bound ribosomes, attached to the cytosolic side of the ER membrane, are engaged in the synthesis of proteins that are being concurrently translocated into the ER. Free ribosomes, unattached to any membrane, synthesize all other proteins encoded by the nuclear genome. Membrane-bound and free ribosomes are structurally and functionally identical. They differ only in the proteins they are making at any given time. When a ribosome happens to be making a protein with an ER signal sequence, the signal directs the ribosome to the ER membrane.

Since many ribosomes can bind to a single mRNA molecule, a polyribosome is usually formed, which becomes attached to the ER membrane, directed there by the signal sequences on multiple growing polypeptide chains (Figure 12-36B). The individual ribosomes associated with such an mRNA molecule can return to the cytosol when they finish translation near the 3′ end of the mRNA molecule. The mRNA itself, however, remains attached to the ER membrane by a changing population of ribosomes, each transiently held at the membrane by the translocator. In contrast, if an mRNA molecule encodes a protein that lacks an ER signal sequence, the polyribosome that forms remains free in the cytosol, and its protein product is discharged there. Therefore, only those mRNA molecules that encode proteins with an ER signal sequence bind to rough ER membranes; those mRNA molecules that encode all other proteins remain free in the cytosol. Individual ribosomal subunits are thought to move randomly between these two segregated populations of mRNA molecules (Figure 12-37).

Smooth ER Is Abundant in Some Specialized Cells

Regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum, or smooth ER. In the great majority of cells, such regions are scanty and are often partly smooth and partly rough. They are sometimes called transitional ER because they contain ER exit sites from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi apparatus. In certain specialized cells, however, the smooth ER is abundant and has additional functions. In particular, it is usually prominent in cells that specialize in lipid metabolism. Cells that synthesize steroid hormones from cholesterol, for example, have an expanded smooth ER compartment to accommodate the enzymes needed to make cholesterol and to modify it to form the hormones (Figure 12-38A).

The main cell type in the liver, the hepatocyte, is another cell with an abundant smooth ER. It is the principal site of production of lipoprotein particles, which carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of lipoproteins are located in the membrane of the smooth ER, which also contains enzymes that catalyze a series of reactions to detoxify both lipid-soluble drugs and various harmful compounds produced by metabolism. The most extensively studied of these detoxification reactions are carried out by the cytochrome P450 family of enzymes, which catalyze a series of reactions in which water-insoluble drugs or metabolites that would otherwise accumulate to toxic levels in cell membranes are rendered sufficiently water-soluble to leave the cell and be excreted in the urine. Because the rough ER alone cannot house enough of these and other necessary enzymes, a major portion of the membrane in a hepatocyte normally consists of smooth ER (Figure 12-38B; see Table 12-2).

When large quantities of certain compounds, such as the drug phenobarbital, enter the circulation, detoxification enzymes are synthesized in the liver in unusually large amounts, and the smooth ER doubles in surface area within a few days. Once the drug has disappeared, the excess smooth ER membrane is specifically and rapidly removed by a lysosome-dependent process called autophagocytosis (discussed in Chapter 13). It is not known how these dramatic changes are regulated.

Another function of the ER in most eucaryotic cells is to sequester Ca²⁺ from the cytosol. The release of Ca²⁺ into the cytosol from the ER, and its subsequent reuptake, is involved in many rapid responses to extracellular signals, as discussed in Chapter 15. The storage of Ca²⁺ in the ER lumen is facilitated by the high concentrations of Ca²⁺-binding proteins there. In some cell types, and perhaps in most, specific regions of the ER are specialized for Ca²⁺ storage. Muscle cells, for example, have an abundant specialized smooth ER, called the sarcoplasmic reticulum, which sequesters Ca²⁺ from the cytosol by means of a Ca²⁺-ATPase that pumps in Ca²⁺ into its lumen. The release and reuptake of Ca²⁺ by the sarcoplasmic reticulum trigger the contraction and relaxation, respectively, of the myofibrils during each round of muscle contraction (discussed in Chapter 16).

We now return to the two major roles of the ER: the synthesis and modification of proteins and the synthesis of lipids.

Rough and Smooth Regions of ER Can Be Separated by Centrifugation

To study the functions and biochemistry of the ER, it is necessary to isolate the ER membrane. This may seem like a hopeless task because the ER is intricately interleaved with other components of the cytosol. Fortunately, when tissues or cells are disrupted by homogenization, the ER breaks into fragments and reseals into many small (~100–200 nm in diameter) closed vesicles called microsomes, which are relatively easy to purify. Microsomes derived from rough ER are studded with ribosomes and are called rough microsomes. The ribosomes are always found on the outside surface, so the interior of the microsome is biochemically equivalent to the lumenal space of the ER (Figure 12-39). Because they can be readily purified in functional form, rough microsomes are especially useful for studying the many processes performed by the rough ER. To the biochemist they represent small authentic versions of the rough ER, still capable of protein synthesis, protein glycosylation, Ca²⁺ uptake, and lipid synthesis.

Many vesicles of a size similar to that of rough microsomes, but lacking attached ribosomes, are also found in these homogenates. Such smooth microsomes are derived in part from smooth portions of the ER and in part from vesiculated fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria (the ratio depending on the tissue). Thus, whereas rough microsomes are derived from rough portions of ER, the origins of smooth microsomes cannot be as easily assigned. The microsomes of the liver are an exception. Because of the unusually large quantities of smooth ER in hepatocytes, most of the smooth microsomes in liver homogenates are derived from smooth ER.

The ribosomes attached to rough microsomes make them more dense than smooth microsomes (Figure 12-39B). As a result, the rough and smooth microsomes can be separated from each other by equilibrium centrifugation (see Figure 12-39A). When the separated rough and smooth microsomes of liver are compared with regard to such properties as enzyme activity or polypeptide composition, they are very similar, although not identical: apparently most of the components of the ER membrane can diffuse freely between the rough and smooth regions, as would be expected for a continuous, fluid membrane. The rough microsomes, however, contain more than 20 proteins that are not present in smooth microsomes, showing that some separation mechanism must exist for a subset of ER membrane proteins. Some of the proteins in this subset help to bind ribosomes to the rough ER, while others presumably produce the flattened shape of this part of the ER (see Figure 12-38B). It is not clear whether these membrane proteins are confined to the rough ER by forming large two-dimensional assemblies in the lipid bilayer, or whether they are instead held in place by interactions with a network of structural proteins on one or the other face of the rough ER membrane.

Signal Sequences Were First Discovered in Proteins Imported into the Rough ER

Signal sequences (and the signal sequence strategy of protein sorting) were first discovered in the early 1970s in secreted proteins that are translocated across the ER membrane as a first step toward their eventual discharge from the cell. In the key experiment, the mRNA encoding a secreted protein was translated by ribosomes in vitro. When microsomes were omitted from this cell-free system, the protein synthesized was slightly larger than the normal secreted protein, the extra length being the N-terminal leader peptide. In the presence of microsomes derived from the rough ER, however, a protein of the correct size was produced. These results were explained by the signal hypothesis, which postulated that the leader serves as an ER signal sequence that directs the secreted protein to the ER membrane and is then cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed (Figure 12-40).

According to the signal hypothesis, the secreted protein should be extruded into the lumen of the microsome during its synthesis in vitro. This can be demonstrated by treatment with a protease: a newly synthesized protein made in the absence of microsomes is degraded when the protease is added to the medium, whereas the same protein made in the presence of microsomes remains intact because it is protected by the microsomal membrane. When proteins without ER signal sequences are similarly synthesized in vitro, they are not imported into microsomes and are therefore degraded by protease treatment.

The signal hypothesis has been thoroughly tested by genetic and biochemical experiments and is found to apply to both plant and animal cells, as well as to protein translocation across the bacterial plasma membrane and, as we have seen, the membranes of mitochondria, chloroplasts, and peroxisomes. N-terminal ER signal sequences guide not only soluble secreted proteins, but also the precursors of all other proteins made by ribosomes bound to the rough ER membrane, including membrane proteins. The signaling function of these peptides has been demonstrated directly by using recombinant DNA techniques to attach ER signal sequences to proteins that do not normally have them; the resulting fusion proteins are directed to the ER.

Cell-free systems in which proteins are imported into microsomes have provided powerful assay procedures for identifying, purifying, and studying the various components of the molecular machinery responsible for the ER import process.

A Signal-Recognition Particle (SRP) Directs ER Signal Sequences to a Specific Receptor in the Rough ER Membrane

The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which cycles between the ER membrane and the cytosol and binds to the signal sequence, and an SRP receptor in the ER membrane. The SRP is a complex particle consisting of six different polypeptide chains bound to a single small RNA molecule (Figure 12-41A). Homologs of the SRP and its receptor are found in all organisms that have been studied, indicating that this protein-targeting mechanism arose early in evolution and has been conserved.

ER signal sequences vary greatly in amino acid sequence, but each has eight or more nonpolar amino acids at its center (see Table 12-3, p. 667). How can the SRP bind specifically to so many different sequences? The answer has come from the crystal structure of the SRP protein, which shows that the signal-sequence-binding site is a large hydrophobic pocket lined by methionines (Figure 12-41B). Because methionines have an unbranched, flexible side chains, the pocket is sufficiently plastic to accommodate hydrophobic signal sequences of different sequences and shapes.

The SRP binds to the ER signal sequence as soon as the peptide has emerged from the ribosome. This causes a pause in protein synthesis, the pause presumably gives the ribosome enough time to bind to the ER membrane before the synthesis of the polypeptide chain is completed, thereby ensuring that the protein is not released into the cytosol. This safety device may be especially important for secreted and lysosomal hydrolases that could wreak havoc in the cytosol; however, cells that secrete large amounts of hydrolases take the added precaution of having high concentrations of hydrolase inhibitors in their cytosol.

Once formed, the SRP-ribosome complex binds to the SRP receptor, which is an integral membrane protein exposed only on the cytosolic surface of the rough ER membrane. This interaction brings the SRP-ribosome complex to a protein translocator. The SRP and SRP receptor are then released, and the growing polypeptide chain is transferred across the membrane (Figure 12-42).

The Polypeptide Chain Passes Through an Aqueous Pore in the Translocator

It has long been debated whether polypeptide chains are transferred across the ER membrane in direct contact with the lipid bilayer or through a pore in a protein translocator. The debate ended with the purification of the protein translocator, which was shown to form a water-filled pore in the membrane through which the polypeptide chain traverses the membrane. The translocator, called the Sec61 complex, consists of three or four protein complexes, each composed of three transmembrane proteins, that assemble into a donutlike structure.

When a ribosome binds, the central hole in the translocator lines up with a tunnel in the large ribosomal subunit through which the growing polypeptide chain exits from the ribosome (Figure 12-43). The bound ribosome forms a tight seal with the translocator, such that the space inside the ribosome is continuous with the lumen of the ER and no molecules can escape from the ER (Figure 12-44). The pore in the translocator cannot be open permanently, however; if it were, Ca²⁺ would leak out of the ER when the ribosome detaches. It is thought that a lumenal ER protein serves as a plug or that the translocator itself can rearrange to close the pore when no ribosome is bound. Thus, the pore is a dynamic structure that opens only transiently when a ribosome with a growing polypeptide chain attaches to the ER membrane.

The signal sequence in the growing polypeptide chain is thought to trigger the opening of the pore: after the signal sequence is released from the SRP and the growing chain has reached a sufficient length, the signal sequence binds to a specific site inside the pore itself, thereby opening the pore. An ER signal sequence is therefore recognized twice: first, by an SRP in the cytosol, and then by a binding site in the ER protein translocator. This may help to ensure that only appropriate proteins enter the lumen of the ER.

Translocation Across the ER Membrane Does Not Always Require Ongoing Polypeptide Chain Elongation

As we have seen, translocation of proteins into mitochondria, chloroplasts, and peroxisomes occurs posttranslationally, after the protein has been made and released into the cytosol, whereas translocation across the ER membrane usually occurs during translation (co-translationally). This explains why ribosomes are bound to the ER but usually not to other organelles.

Some proteins, however, are imported into the ER after their synthesis has been completed, demonstrating that translocation does not always require ongoing translation. Posttranslational protein translocation is especially common across the ER membrane in yeast cells and across the bacterial plasma membrane (which is thought to be evolutionarily related to the ER; see Figure 12-4). To function in posttranslational translocation, the translocator needs accessory proteins that feed the polypeptide chain into the pore and drive translocation (Figure 12-45). In bacteria, a translocation motor protein, the SecA ATPase, attaches to the cytosolic side of the translocator, where it undergoes cyclic conformational changes driven by ATP hydrolysis. Each time an ATP is hydrolyzed, a portion of the SecA protein inserts into the pore of the translocator, pushing a short segment of the passenger protein with it. As a result of this ratchet mechanism, the SecA protein pushes the polypeptide chain of the transported protein across the membrane.

Eucaryotic cells use a different set of accessory proteins that associate with the Sec61 complex. These proteins span the ER membrane and use a small domain on the lumenal side of the ER membrane to deposit an hsp70-like chaperone protein (called BiP, for binding protein) onto the polypeptide chain as it emerges from the pore into the ER lumen. Unidirectional translocation is driven by cycles of BiP binding and release, as described earlier for the mitochondrial hsp70 proteins that pull proteins across mitochondrial membranes.

Proteins that are transported into the ER by a posttranslational mechanism are first released into the cytosol, where they are prevented from folding up by binding to chaperone proteins, as discussed earlier for proteins destined for mitochondria and chloroplasts. In all of these cases where translocation occurs without a ribosome sealing the pore, it remains a mystery how the polypeptide chain can slide through the pore in the translocator without allowing ions and other molecules to pass through.

The ER Signal Sequence Is Removed from Most Soluble Proteins After Translocation

We have seen that in chloroplasts and mitochondria, the signal sequence is cleaved from precursor proteins once it has crossed the membrane. Similarly, N-terminal ER signal sequences are removed by a signal peptidase on the lumenal side of the ER membrane. The signal sequence by itself, however, is not sufficient for signal cleavage by the peptidase; this requires an adjacent cleavage site that is specifically recognized by the peptidase. We shall see below that ER signal sequences that occur within the polypeptide chain—rather than at the N-terminus—do not have these recognition sites and are never cleaved; instead, they can serve to retain transmembrane proteins in the lipid bilayer after the translocation process has been completed.

The N-terminal ER signal sequence of a soluble protein has two signaling functions. It directs the protein to the ER membrane, and it serves as a start-transfer signal (or start-transfer peptide) that opens the pore. Even after it is cleaved off by signal peptidase, the signal sequence is thought to remain bound to the translocator while the rest of the protein is threaded continuously through the membrane as a large loop. Once the C-terminus of the protein has passed through the membrane, the translocated protein is released into the ER lumen (Figure 12-46). The signal sequence is released from the pore and rapidly degraded to amino acids by other proteases in the ER.

While bound in the translocation pore, signal sequences are in contact not only with the Sec61 complex, which forms the walls of the pore, but also with the hydrophobic lipid core of the membrane. This was shown in chemical cross-linking experiments in which signal sequences and the hydrocarbon chains of lipids could be covalently linked together. To release the signal sequence into the membrane, the translocator has to open laterally. The translocator is therefore gated in two directions: it can open to form a pore across the membrane to let the hydrophilic portions of proteins cross the lipid bilayer, and it can open laterally within the membrane to let hydrophobic portions of proteins partition into the bilayer. This lateral gating mechanism is crucial for the insertion of transmembrane proteins into the lipid bilayer, as we discuss next.

In Single-Pass Transmembrane Proteins, a Single Internal ER Signal Sequence Remains in the Lipid Bilayer as a Membrane-spanning α Helix

The translocation process for proteins destined to remain in the membrane is more complex than it is for soluble proteins, as some parts of the polypeptide chain are translocated across the lipid bilayer whereas others are not. Nevertheless, all modes of insertion of membrane proteins can be considered as variants of the sequence of events just described for transferring a soluble protein into the lumen of the ER. We begin by describing the three ways in which single-pass transmembrane proteins (see Figure 10-17) become inserted into the ER.

In the simplest case, an N-terminal signal sequence initiates translocation, just as for a soluble protein, but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is translocated. This stop-transfer signal anchors the protein in the membrane after the ER signal sequence (the start-transfer signal) has been released from the translocator and has been cleaved off (Figure 12-47). The stop-transfer sequence is transferred into the bilayer by the lateral gating mechanism, and it remains there as a single α-helical membrane-spanning segment, with the N-terminus of the protein on the lumenal side of the membrane and the C-terminus on the cytosolic side.

In the other two cases, the signal sequence is internal, rather than at the N-terminal end of the protein. Like the N-terminal ER signal sequences, the internal signal sequence is recognized by an SRP, which brings the ribosome making the protein to the ER membrane and serves as a start-transfer signal that initiates the translocation of the protein. After release from the translocator, the internal start-transfer sequence remains in the lipid bilayer as a single membrane-spanning α helix.

Internal start-transfer sequences, can bind to the translocation apparatus in either of two orientations, and the orientation of the inserted start-transfer sequence, in turn, determines which protein segment (the one preceding or the one following the start-transfer sequence) is moved across the membrane into the ER lumen. In one case, the resulting membrane protein has its C-terminus on the lumenal side (Figure 12-48A), while in the other, it has its N-terminus on the lumenal side (Figure 12-48B). The orientation of the start-transfer sequence depends on the distribution of nearby charged amino acids, as described in the figure legend.

Combinations of Start-Transfer and Stop-Transfer Signals Determine the Topology of Multipass Transmembrane Proteins

In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer (see Figure 10-17). It is thought that an internal signal sequence serves as a start-transfer signal in these proteins to initiate translocation, which continues until a stop-transfer sequence is reached. In double-pass transmembrane proteins, for example, the polypeptide can then be released into the bilayer (Figure 12-49). In more complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptide release, and so on for subsequent start-transfer and stop-transfer sequences (Figure 12-50).

Whether a given hydrophobic signal sequence functions as a start-transfer or stop-transfer sequence must depend on its location in a polypeptide chain, since its function can be switched by changing its location in the protein using recombinant DNA techniques. Thus, the distinction between start-transfer and stop-transfer sequences results mostly from their relative order in the growing polypeptide chain. It seems that the SRP begins scanning an unfolded polypeptide chain for hydrophobic segments at its N-terminus and proceeds toward the C-terminus, in the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame”: if translocation is initiated, the next appropriate hydrophobic segment is recognized as a stop-transfer sequence, causing the region of the polypeptide chain in between to be threaded across the membrane. A similar scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane.

Because membrane proteins are always inserted from the cytosolic side of the ER in this programmed manner, all copies of the same polypeptide chain will have the same orientation in the lipid bilayer. This generates an asymmetrical ER membrane in which the protein domains exposed on one side are different from those domains exposed on the other. This asymmetry is maintained during the many membrane budding and fusion events that transport the proteins made in the ER to other cell membranes (discussed in Chapter 13). Thus, the way in which a newly synthesized protein is inserted into the ER membrane determines the orientation of the protein in all of the other membranes as well.

When proteins are dissociated from a membrane and are then reconstituted into artificial lipid vesicles, a random mixture of right-side-out and inside-out protein orientations usually results. Thus, the protein asymmetry observed in cell membranes seems not to be an inherent property of the protein, but instead results solely from the process by which proteins are inserted into the ER membrane from the cytosol.

Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER

Many of the proteins in the lumen of the ER are in transit, en route to other destinations; others, however, are normally resident there and are present at high concentrations. These ER resident proteins contain an ER retention signal of four amino acids at their C terminus that is responsible for retaining the protein in the ER (see Table 12-3; discussed in Chapter 13). Some of these proteins function as catalysts that help the many proteins that are translocated into the ER to fold and assemble correctly.

One important ER resident protein is protein disulfide isomerase (PDI), which catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S-S) bonds. Almost all cysteines in protein domains exposed to either the extracellular space or the lumen of organelles in the secretory and endocytic pathways are disulfide-bonded; disulfide bonds do not form, however, in domains exposed to the cytosol because of the reducing environment there.

Another ER resident protein is the chaperone protein BiP. We have already discussed how BiP works to pull proteins posttranslationally into the ER through the ER translocator. Like other chaperones, BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. To do so, it binds to exposed amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains. An example of a BiP-binding site is a stretch of alternating hydrophobic and hydrophilic amino acids that would normally be buried in a β sheet. The bound BiP both prevents the protein from aggregating and helps to keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). Like the hsp70 family of proteins, which bind unfolded proteins in the cytosol and facilitate their import into mitochondria and chloroplasts, BiP hydrolyzes ATP to provide the energy for its roles in protein folding and posttranslational import into the ER.

Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a Common N-linked Oligosaccharide

The covalent addition of sugars to proteins is one of the major biosynthetic functions of the ER. Most of the soluble and membrane-bound proteins that are made in the ER—including those destined for transport to the Golgi apparatus, lysosomes, plasma membrane, or extracellular space—are glycoproteins. In contrast, very few proteins in the cytosol are glycosylated, and those that are carry a much simpler sugar modification, in which a single N-acetylglucosamine group is added to a serine or threonine residue of the protein.

An important advance in understanding the process of protein glycosylation was the discovery that a preformed precursor oligosaccharide (composed of N-acetylglucosamine, mannose, and glucose and containing a total of 14 sugars) is transferred en bloc to proteins in the ER. Because this oligosaccharide is transferred to the side-chain NH₂ group of an asparagine amino acid in the protein, it is said to be N-linked or asparagine-linked (Figure 12-51). The transfer is catalyzed by a membrane-bound enzyme, an oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. The precursor oligosaccharide is held in the ER membrane by a special lipid molecule called dolichol, and it is transferred to the target asparagine in a single enzymatic step immediately after that amino acid has emerged into the ER lumen during protein translocation (Figure 12-52). Since most proteins are co-translationally imported into the ER, N-linked oligosaccharides are almost always added during protein synthesis.

The precursor oligosaccharide is linked to the dolichol lipid by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction illustrated in Figure 12-52. The entire precursor oligosaccharide is built up sugar by sugar on this membrane-bound lipid molecule before its transfer to a protein. The sugars are first activated in the cytosol by the formation of nucleotide-sugar intermediates, which then donate their sugar (directly or indirectly) to the lipid in an orderly sequence. Partway through this process, the lipid-linked oligosaccharide is flipped from the cytosolic to the lumenal side of the ER membrane (Figure 12-53).

All of the diversity of the N-linked oligosaccharide structures on mature glycoproteins results from the later modification of the original precursor oligosaccharide. While still in the ER, three glucoses (see Figure 12-51) and one mannose are quickly removed from the oligosaccharides of most glycoproteins. We shall return to the importance of glucose trimming shortly. This oligosaccharide “trimming” or “processing” continues in the Golgi apparatus and is discussed in Chapter 13.

The N-linked oligosaccharides are by far the most common oligosaccharides found on glycoproteins. Less frequently, oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine, or hydroxylysine amino acid. These O-linked oligosaccharides are formed in the Golgi apparatus by pathways that are not yet fully understood.

Oligosaccharides Are Used as Tags to Mark the State of Protein Folding

It has long been debated why glycosylation is such a common modification of proteins that enter the ER. One particularly puzzling observation has been that some proteins require N-linked glycosylation for proper folding in the ER, yet the precise location of the oligosaccharides attached to the protein's surface does not seem to matter. A clue to the role of glycosylation in protein folding came from studies of two ER chaperone proteins that are called calnexin and calreticulin because they require Ca²⁺ for their activities. These chaperones are lectins that bind to oligosaccharides on incompletely folded proteins and retain them in the ER. Like other chaperones, they prevent incompletely folded proteins from undergoing irreversible aggregation. Both calnexin and calreticulin also promote the association of incompletely folded protein with another ER chaperone, which binds to cysteines that have not yet formed disulfide bonds.

Calnexin and calreticulin recognize N-linked oligosaccharides that contain a single terminal glucose, and therefore bind proteins only after two of the three glucoses that are initially attached have been removed by ER glucosidases. When the third glucose is removed, the protein dissociates from its chaperone and can leave the ER.

How, then, do calnexin and calreticulin distinguish folded from incompletely folded proteins? The answer lies in yet another ER enzyme, a glucosyl transferase that keeps adding a glucose to those oligosaccharides that have lost their last glucose. It adds the glucose, however, only to oligosaccharides that are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous cycles of glucose trimming (by glucosidase) and addition (by glycosyl transferase), and maintains an affinity for calnexin and calreticulin until it has achieved its fully folded state (Figure 12-54).

Improperly Folded Proteins Are Exported from the ER and Degraded in the Cytosol

Despite all the help from chaperones, many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve their properly folded or oligomeric state. Such proteins are exported from the ER back into the cytosol, where they are degraded. The retrotranslocation, also called dislocation, occurs via the same translocator (the Sec61 complex) through which the proteins entered the ER in the first place, although additional proteins help the translocator to function in reverse. It is not known how such misfolded proteins, which no longer have their ER signal sequences, are recognized or transferred.

Once the misfolded protein has reached the cytosol, its oligosaccharides are removed. Deglycosylation is catalyzed by an N-glycanase, which removes the oligosaccharide chains by cleaving the amide bond between the carbonyl group and the amino group of the original asparagine to which the oligosaccharide was attached. The deglycosylated polypeptide is rapidly ubiquitylated by ER-bound ubiquitin-conjugating enzymes and is then fed into proteasomes (discussed in Chapter 6), where it is degraded (Figure 12-55).

Misfolded Proteins in the ER Activate an Unfolded Protein Response

Cells carefully monitor the amount of misfolded proteins they contain in various compartments. An accumulation of misfolded proteins in the cytosol, for example, triggers a heat-shock response (discussed in Chapter 6), which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins. Similarly, an accumulation of misfolded proteins in the ER triggers an unfolded protein response, which includes an increased transcription of genes encoding ER chaperones and enzymes involved in ER protein degradation.

How do misfolded proteins in the cytosol or ER signal to the nucleus? The pathway from the ER to the nucleus is especially well understood in yeast cells, and it is remarkable. A transmembrane protein kinase in the ER is activated by misfolded proteins, which cause its oligomerization and autophosphorylation. (Extracellular growth factors activate their receptors in the plasma membrane in a similar way, as discussed in Chapter 15). Oligomerization of the ER kinase leads to the activation of an endoribonuclease domain contained on the same molecule. This nuclease cleaves a specific, cytosolic RNA molecule at two positions, excising an intron. The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated on ribosomes to produce a gene regulatory protein. The protein migrates to the nucleus and activates the transcription of the genes encoding the proteins that mediate the unfolded protein response (Figure 12-56).

Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor

As discussed in Chapter 10, several cytosolic enzymes catalyze the covalent addition of a single fatty acid chain or prenyl group to selected proteins. The attached lipids help to direct these proteins to cell membranes. A related process is catalyzed by ER enzymes, which covalently attach a glycosylphosphatidyl-inositol (GPI) anchor to the C terminus of some membrane proteins destined for the plasma membrane. This linkage forms in the lumen of the ER, where, at the same time, the transmembrane segment of the protein is cleaved off (Figure 12-57). A large number of plasma membrane proteins are modified in this way. Since they are attached to the exterior of the plasma membrane only by their GPI anchors, they can in principle be released from cells in soluble form in response to signals that activate a specific phospholipase in the plasma membrane. Trypanosome parasites, for example, use this mechanism to shed their coat of GPI-anchored surface proteins if attacked by the immune system. GPI anchors are also used to direct plasma membrane proteins into lipid rafts and thus segregate the proteins from other membrane proteins, as we discuss in Chapter 13.

Most Membrane Lipid Bilayers Are Assembled in the ER

The ER membrane synthesizes nearly all of the major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The major phospholipid made is phosphatidylcholine (also called lecithin), which can be formed in three steps from choline, two fatty acids, and glycerol phosphate (Figure 12-58). Each step is catalyzed by enzymes in the ER membrane that have their active sites facing the cytosol, where all of the required metabolites are found. Thus, phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane. In the first step, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid, a compound sufficiently water-insoluble to remain in the lipid bilayer after it has been synthesized. It is this step that enlarges the lipid bilayer. The later steps determine the head group of a newly formed lipid molecule, and therefore the chemical nature of the bilayer, but they do not result in net membrane growth. The two other major membrane phospholipids—phosphatidyl-ethanolamine and phosphatidylserine—as well as the minor phospholipid phosphatidylinositol (PI), are all synthesized in this way.

Because phospholipid synthesis takes place in the cytosolic half of the ER bilayer, there needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer. In synthetic lipid bilayers, lipids do not “flip-flop” in this way. In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flip-flop.” This rapid trans-bilayer movement is thought to be mediated by a phospholipid translocator called a scramblase that equilibrates phospholipids between the two leaflets of the lipid bilayer (Figure 12-59). Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane. The plasma membrane contains, in addition to the scramblase, a different type of phospholipid translocator that belongs to the family of ABC transporters (discussed in Chapter 11). These flippases specifically remove phospholipids containing free amino groups (phosphatidylserine and phosphatidylethanolamine) from the extracellular leaflet and use the energy of ATP hydrolysis to flip them directionally into the leaflet facing the cytosol. The plasma membrane therefore has a highly asymmetric phospholipid composition, which is actively maintained by the flippases (see Figure 10-14).

The ER also produces cholesterol and ceramide. Ceramide is made by condensing the amino acid serine with a fatty acid to form the amino alcohol sphingosine; a second fatty acid is then added to form ceramide. The ceramide is exported to the Golgi apparatus, where it serves as a precursor for the synthesis of two types of lipids: oligosaccharide chains are added to form glycosphingo-lipids (glycolipids), and phosphocholine head groups are transferred from phosphatidylcholine to other ceramide molecules to form sphingomyelin. Thus, both glycolipids and sphingomyelin are produced relatively late in the process of membrane synthesis. Because they are produced by enzymes exposed to the Golgi lumen and are not substrates for lipid translocators, they are found exclusively in the noncytosolic leaflet of the lipid bilayers that contain them.

Phospholipid Exchange Proteins Help to Transport Phospholipids from the ER to Mitochondria and Peroxisomes

As discussed in Chapter 13, the plasma membrane and the membranes of the Golgi apparatus, lysosomes, and endosomes all form part of a membrane system that communicates with the ER by means of transport vesicles that transfer both proteins and lipids. Mitochondria, plastids, and possibly peroxisomes, however, do not belong to this system, and they therefore require different mechanisms for the import of proteins and lipids for growth. We have already seen that most of the proteins in these organelles are imported from the cytosol. Although mitochondria modify some of the lipids they import, they do not synthesize lipids from scratch; instead, their lipids have to be imported from the ER, either directly, or indirectly by way of other cell membranes. In either case, special mechanisms are required for the transfer.

Water-soluble carrier proteins called phospholipid exchange proteins (or phospholipid transfer proteins) transfer individual phospholipid molecules between membranes. Each exchange protein recognizes only specific types of phospholipids. It functions by “extracting” a molecule of the appropriate phospholipid from a membrane and diffusing away with the lipid buried within its lipid-binding site. When it encounters another membrane, the exchange protein tends to discharge the bound phospholipid molecule into the new lipid bilayer (Figure 12-60). It has been proposed that phosphatidylserine is imported into mitochondria in this way, where it is then decarboxylated to yield phosphatidylethanolamine. Phosphatidylcholine, by contrast, is imported intact.

Exchange proteins act to distribute phospholipids at random between all membranes present. In principle, such a random exchange process can result in a net transport of lipids from a lipid-rich to a lipid-poor membrane, allowing phosphatidylcholine and phosphatidylserine molecules, for example, to be transferred from the ER, where they are synthesized, to a mitochondrial or peroxisomal membrane. It might be that mitochondria and peroxisomes are the only “lipid-poor” organelles in the cytosol and that such an exchange process is sufficient. In electron micrographs, mitochondria are often seen in close juxtaposition to ER membranes, and there may be specific mechanisms of lipid transfer that operate at such regions of proximity.

Summary

The extensive ER network serves as a factory for the production of almost all of the cell's lipids. In addition, a major portion of the cell's protein synthesis occurs on the cytosolic surface of the ER: all proteins destined for secretion and all proteins destined for the ER itself, the Golgi apparatus, the lysosomes, the endosomes, and the plasma membrane are first imported into the ER from the cytosol. In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and N-linked oligosaccharides are added. N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate excessively in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to help the ER to cope.

Only proteins that carry a special ER signal sequence are imported into the ER. The signal sequence is recognized by a signal recognition particle (SRP), which binds both the growing polypeptide chain and a ribosome and directs them to a receptor protein on the cytosolic surface of the rough ER membrane. This binding to the ER membrane initiates the translocation process by threading a loop of polypeptide chain across the ER membrane through the hydrophilic pore in a transmembrane protein translocator.

Soluble proteins—destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles—pass completely into the ER lumen. Transmembrane proteins destined for the ER or for other cell membranes are translocated partway across the ER membrane and remain anchored there by one or more membrane-spanning α-helical regions in their polypeptide chains. These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during the translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein.

The asymmetry of protein insertion and glycosylation in the ER establishes the sidedness of the membranes of all of the other organelles that the ER supplies with membrane proteins.