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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

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Molecular Biology of the Cell. 4th edition.

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The Transport of Proteins into Mitochondria and Chloroplasts

As discussed in Chapter 14, mitochondria and chloroplasts are double-membrane-enclosed organelles. They specialize in the synthesis of ATP, using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthesis in chloroplasts. Although both organelles contain their own DNA, ribosomes, and other components required for protein synthesis, most of their proteins are encoded in the cell nucleus and imported from the cytosol. Moreover, each imported protein must reach the particular organelle subcompartment in which it functions.

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There are two subcompartments in mitochondria: the internal matrix space and the intermembrane space. These compartments are formed by the two concentric mitochondrial membranes: the inner membrane, which forms extensive invaginations, the cristae, and encloses the matrix space, and the outer membrane, which is in contact with the cytosol (Figure 12-22A). Chloroplasts have the same two subcompartments plus an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane (Figure 12-22B). Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins.

Figure 12-22. The subcompartments of mitochondria and chloroplasts.

Figure 12-22

The subcompartments of mitochondria and chloroplasts. In contrast to the cristae of mitochondria (A), the thylakoids of chloroplasts (B) are not connected to the inner membrane and therefore form a compartment with a separate internal space (see Figure (more...)

New mitochondria and chloroplasts are produced by the growth of preexisting organelles followed by fission (discussed in Chapter 14). Their growth depends mainly on the import of proteins from the cytosol. This requires that proteins be translocated across a number of membranes in succession and end up in the appropriate place. How this occurs is the subject of this section.

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.

Figure 12-23. A signal sequence for mitochondrial protein import.

Figure 12-23

A signal sequence for mitochondrial protein import. Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain (discussed in Chapter 14). (A) (more...)

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.

Figure 12-24. Three protein translocators in the mitochondrial membranes.

Figure 12-24

Three protein translocators in the mitochondrial membranes. The TOM and TIM complexes and the OXA complex are multimeric membrane protein assemblies that catalyze protein transport across mitochondrial membranes. The protein components of the TIM22 and (more...)

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.

Figure 12-25. Proteins transiently spanning the inner and outer mitochondrial membranes during their translocation into the matrix.

Figure 12-25

Proteins transiently spanning the inner and outer mitochondrial membranes during their translocation into the matrix. When isolated mitochondria are incubated with a precursor protein at 5°C, the precursor is only partly translocated. The N-terminal (more...)

Figure 12-26. Protein import by mitochondria.

Figure 12-26

Protein import by mitochondria. The N-terminal signal sequence of the precursor protein is recognized by receptors of the TOM complex. The protein is thought to be translocated across both mitochondrial membranes at or near special contact sites. The (more...)

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.

Figure 12-27. The role of energy in protein import into the mitochondrial matrix.

Figure 12-27

The role of energy in protein import into the mitochondrial matrix. (1) Bound cytosolic hsp70 is released from the protein in a step that depends on ATP hydrolysis. After initial insertion of the signal sequence and of adjacent portions of the polypeptide (more...)

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.

Figure 12-28. Two plausible models of how mitochondrial hsp70 could drive protein import.

Figure 12-28

Two plausible models of how mitochondrial hsp70 could drive protein import. (A) In the thermal ratchet model, the translocating polypeptide chain slides back and forth, driven by thermal motion, and it is successively trapped in the matrix by hsp70 binding. (more...)

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.

Figure 12-29. Protein import from the cytosol into the inner mitochondrial membrane or intermembrane space.

Figure 12-29

Protein import from the cytosol into the inner mitochondrial membrane or intermembrane space. (A) A pathway that requires two signal sequences and two translocation events is thought to be used to move some proteins from the cytosol to the inner membrane. (more...)

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).

Figure 12-30. Translocation of a precursor protein into the thylakoid space of chloroplasts.

Figure 12-30

Translocation of a precursor protein into the thylakoid space of chloroplasts. (A) The precursor protein contains an N-terminal chloroplast signal sequence (red), followed immediately by a thylakoid signal sequence (orange). The chloroplast signal sequence (more...)


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.

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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
Bookshelf ID: NBK26828