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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 17.1Synthesis and Targeting of Mitochondrial and Chloroplast Proteins

Mitochondria and chloroplasts are surprisingly similar. Both are bounded by two membranes; chloroplasts contain, in addition, an internal membrane compartment — the thylakoids — on which photosynthesis takes place (see Figure 16-34). Both organelles use a proton-motive force and the same type of protein — an F-class ATPase — to synthesize ATP (see Figure 16-2); they also contain similar types of electron-transport proteins. Growth and division of mitochondria and chloroplasts is not coupled to nuclear division. These organelles grow by the incorporation of proteins and lipids, a process that occurs continuously during the interphase period of the cell cycle. As the organelles increase in size, one or more daughters pinch off in a manner similar to the way in which bacterial cells grow and divide. Although the biogenesis of both organelles is similar in many respects, our discussion focuses on mitochondrial biogenesis, about which more is known.

Mitochondria and chloroplasts probably arose by the incorporation of photosynthetic or nonphotosynthetic bacteria into ancestral eukaryotic cells, about 1,500 million years ago, and their subsequent replication in the cytoplasm. Over eons of evolution much of the bacterial DNA in these endosymbionts moved to the nucleus, so that in present-day cells many mitochondrial and chloroplast proteins are imported into the organelles after their synthesis in the cytosol. The mitochondrial and chloroplast DNA found in extant organisms encodes organelle rRNAs and tRNAs but relatively few proteins, mainly subunits of integral membrane proteins essential to organelle function (see Figure 9-44). These proteins are synthesized on ribosomes within the organelles and directed to the correct compartment immediately after synthesis, and current work is elucidating how this happens.

Most chloroplast and mitochondrial proteins, however, are synthesized outside the organelle on cytosolic ribosomes that are not bound to the rough endoplasmic reticulum. The newly made proteins are released into the cytosol and are then taken up specifically into the proper organelle by binding to receptor proteins on the organelle surface that recognize specific uptake-targeting sequences in the new proteins (Table 17-1). The mitochondrion and chloroplast contain multiple membranes and membrane-limited spaces. Thus targeting of some proteins requires the sequential action of two targeting sequences and two membrane-bound receptor systems: one to direct the protein into the organelle, and the other to direct it into the correct organellar compartment or membrane (see Figure 17-1). In general, protein uptake into mitochondria or chloroplasts is an energy-requiring process that depends on integral proteins in the organellar membranes. The biogenesis of both organelles is similar in many respects.

Table 17-1. Properties of Uptake-Targeting Signal Sequences That Direct Proteins from the Cytosol to Organelles.

Table 17-1

Properties of Uptake-Targeting Signal Sequences That Direct Proteins from the Cytosol to Organelles.

Most Mitochondrial Proteins Are Synthesized as Cytosolic Precursors Containing Uptake-Targeting Sequences

Some of the proteins that are synthesized in the cytosol and incorporated into the mitochondrion are listed in Table 17-2. The largest number are transported to the matrix, but some are transported to the intermembrane space or inserted into the outer or inner membrane. Pulse-chase experiments with yeast and Neurospora cells clearly demonstrate that proteins made in the cytosol can be incorporated into mitochondria. When these cells are treated with a radiolabeled amino acid (e.g., [35S]methionine) for a few seconds (the “pulse” label), all newly made mitochondrial proteins are initially located in the cytosol outside the mitochondria. The incorporation of radioactivity is then blocked by incubating the cells with a drug that inhibits protein synthesis or with abundant unlabeled methionine (the “chase” period); subsequently, the radiolabeled proteins in the cytosol gradually accumulate at their proper destinations in the mitochondrion.

Table 17-2. Selected Mitochondrial Proteins That Are Synthesized in the Cytosol.

Table 17-2

Selected Mitochondrial Proteins That Are Synthesized in the Cytosol.

Furthermore, most proteins imported into the mitochon-drion begin as precursors containing amino acids at the N-terminus that are not present in the mature protein. These N-terminal residues comprise one or more targeting sequences that direct the protein to its proper destination within the mitochondrion (Figure 17-2). Mitochondrial precursor proteins can be synthesized in cell-free systems in the absence of mitochondria. When mitochondria are subsequently added, the precursors are incorporated into the organelle and in most cases, the N-terminal uptake-targeting sequences are removed (Figure 17-3).

Figure 17-2. Uptake-targeting sequences of imported mitochondrial proteins.

Figure 17-2

Uptake-targeting sequences of imported mitochondrial proteins. All such proteins have an N-terminal matrix-targeting sequence (red) that is similar but not identical in different proteins. Proteins destined for the intermembrane space and outer membrane (more...)

Figure 17-3. Cell-free system for studying post-translational uptake of mitochondrial proteins.

Figure 17-3

Cell-free system for studying post-translational uptake of mitochondrial proteins. Precursor proteins with an N-terminal uptaketargeting sequence can be produced in an in vitro protein-synthesizing system from the corresponding mRNAs (step 1 ). Protein (more...)

All proteins that travel from the cytosol to the same mitochondrial destination have targeting signals that share common motifs, although the signal sequences are generally not identical (see Table 17-1 and Figure 17-2). Thus, the receptors that recognize such sequences, including the mitochondrial outer-membrane receptors that bind matrix-targeting sequences, are able to bind to a number of different but related sequences. The matrix-targeting sequences of the various mitochondrial proteins are rich in positively charged amino acids — arginine and lysine — and hydroxylated ones  — serine and threonine; they also are devoid of the acidic residues aspartate and glutamate. These sequences contain all the information required to target precursor proteins from the cytosol to the mitochondrial matrix.

Cytosolic Chaperones Deliver Proteins to Channel-Linked Receptors in the Mitochondrial Membrane

Figure 17-4 presents an overview of protein import from the cytosol into the mitochondrial matrix, the route into the mitochondrion followed by most imported proteins. We will discuss in detail each step in protein transport into the matrix and then consider how proteins are targeted to other compartments of the mitochondrion.

Figure 17-4. Protein import into the mitochondrial matrix.

Figure 17-4

Protein import into the mitochondrial matrix. As a precursor protein, with its N-terminal matrix-targeting sequence (red), emerges from cytosolic ribosomes, it binds to chaperone proteins, such as mitochondrial-import stimulating factor (MSF) and cytosolic (more...)

In the cytosol, the soluble precursors of mitochondrial proteins (including hydrophobic integral membrane proteins) bind to one or more chaperones. These proteins use the energy released by ATP hydrolysis to keep nascent and newly made proteins in an unfolded state (see Figure 3-15). Two chaperones, cytosolic Hsc70 and mitochondrial-import stimulation factor (MSF), have been shown to prevent the misfolding or aggregation of mitochondrial precursor proteins so that they can be taken up by mitochondria. MSF is also able to disperse aggregates of proteins.

Some precursor proteins, such as the inner-membrane ATP/ADP antiporter, bind to MSF and the resulting complex then binds to a set of receptors called Tom37 and Tom70 on the outer membrane; Tom37 and Tom70 then transfer the precursor to a second set of receptors (Tom20 and Tom22) with release of MSF (Figure 17-4, steps 1a, 2, and 3a). Most precursor proteins bind to cytosolic Hsc70, which delivers the protein directly to the Tom20 and Tom22 receptors (steps 1b and 3b). These receptors are linked to Tom40, the actual channel in the outer membrane. When purified and incorporated into liposomes, Tom40 forms a transmembrane channel with a pore wide enough — about 1.5 to 2.5 nm in diameter — to accommodate an unfolded polypeptide chain.

A precursor protein destined for the mitochondrial matrix passes through the Tom40 channel in the outer membrane and another channel in the inner membrane (Figure 17-4, steps 4 and 5). Translocation into the matrix occurs only at “contact sites” where the outer and inner membranes are in close proximity and requires a proton-motive force across the inner membrane.

Matrix Chaperones and Chaperonins Are Essential for the Import and Folding of Mitochondrial Proteins

As proteins enter the mitochondrial matrix, they are bound by matrix Hsc70, a chaperone similar in structure and function to cytosolic Hsc70 (see Figure 3-15). Matrix Hsc70 is bound to the matrix surface of the inner mitochondrial membrane close to the channels traversed by imported proteins. Like cytosolic Hsc70, matrix Hsc70 binds the unfolded form of imported proteins as they emerge into the matrix, preventing protein aggregation or precipitation and premature folding (Figure 17-4, step 5). This is particularly important for subunits of multiprotein complexes, since the proper folding of any one subunit requires the presence of all of the protein’s subunits. The energy released by Hsc70-catalyzed ATP hydrolysis also may help power translocation of proteins into the matrix.

Soon after a protein arrives in the mitochondrial matrix, a protease removes its N-terminal matrix-targeting sequence (Figure 17-4, step 6). During in vitro reactions, this matrix enzyme, a dimeric metal-containing protease, specifically cleaves the N-terminal matrix-targeting sequence from several different precursor proteins.

Some imported proteins can fold into their final, active conformation without further assistance (Figure 17-4, step 7a). Final folding of many matrix proteins, however, requires Hsc60, a matrix chaperonin (step 7b). Chaperonin proteins such as bacterial GroEL, to which Hsc60 is related, actively facilitate protein folding (see Figure 3-15). Evidence for the importance of Hsc60 in folding of imported mitochondrial proteins comes from yeast mutants defective in this chaperonin. In these mutants, import of matrix proteins (e.g., the β subunit of the F1 ATPase) and cleavage of the uptake-targeting sequence occur normally, but the imported polypeptide fails to assemble into a normal multiprotein complex.

Studies with Chimeric Proteins Confirm Major Features of Mitochondrial Import

Three properties characterize protein import into mitochondria: (1) all the information required to target a precursor protein from the cytosol to the mitochondrial matrix is contained within its N-terminal matrix-targeting sequence; (2) only unfolded proteins can be imported; and (3) translocation of precursors to the matrix occurs at the rare sites where the outer and inner membranes are close together.

Dramatic evidence for the ability of matrix-targeting sequences to direct import was obtained with chimeric proteins produced by recombinant DNA techniques. For example, the matrix-targeting sequence of alcohol dehydrogenase can be fused to the N-terminus of dihydrofolate reductase (DHFR), which normally resides in the cytosol (Figure 17-5, top). In the presence of chaperones, which prevent the C-terminal DHFR segment from folding in the cytosol, the chimeric protein is transported into the matrix of energized mitochondria, and the targeting sequence is cleaved normally (Figure 17-5a). Conversely, deleting or mutating its matrix- targeting sequence causes a protein normally transported to the matrix to remain in the cytosol.

Figure 17-5. Experiments with chimeric proteins elucidate mitochondrial protein import.

Figure 17-5

Experiments with chimeric proteins elucidate mitochondrial protein import. (Top) The chimeric protein used contained at its N-terminus a matrix-targeting signal, followed by a spacer sequence of no particular function, and then by dihydrofolate reductase (more...)

Binding of the DHFR inhibitor methotrexate to the active site on the DHFR segment of the chimera causes it to become locked in a folded state. The finding that the chimeric protein does not cross into the matrix in the presence of methotrexate indicates that only unfolded proteins can enter the matrix. However, the N-terminal matrix-targeting sequence of the chimera still enters the matrix space, where it is cleaved, leaving the rest of the chimera stuck in the membrane as a stable translocation intermediate (Figure 17-5b). The chimeric protein in this experiment contains a spacer sequence of 50 amino acids between the targeting sequence and DHFR segment. In the translocation intermediate, the spacer sequence spans both membranes; it is long enough to do so only if it is in an extended conformation, stretched to its maximum possible length. If the chimera contains a shorter spacer — say 35 amino acids — no stable translocation intermediate is obtained, because the spacer can not traverse both membranes. These observations provide further evidence that translocated proteins traverse the membrane in an unfolded state.

Microscopic studies of stable translocation intermediates show that they accumulate at sites where the inner and outer mitochondrial membranes are close together, evidence that precursor proteins enter only at such sites (Figure 17-5c). Outer-membrane receptors and other components of the mitochondrial protein-importing machinery are localized at or near these contact sites. Moreover, stable translocation intermediates can be chemically cross-linked to integral proteins of both the outer and inner membranes, strongly suggesting that imported proteins traverse both the outer and inner mitochondrial membranes in protein-lined channels as depicted in Figure 17-4. Since ≈1000 stuck translocation intermediates can be observed in a typical yeast mitochondrion, it is thought that mitochondria have ≈1000 transport channels for the uptake of mitochondrial proteins.

The Uptake of Mitochondrial Proteins Requires Energy

Studies on import of mitochondrial proteins in cell-free systems have shown that three separate inputs of energy are required: ATP hydrolysis in the cytosol, a proton-motive force across the inner membrane, and ATP hydrolysis in the mitochondrial matrix (see Figure 17-4).

As noted earlier, cytosolic chaperone proteins bound to precursor mitochondrial proteins couple the energy released by ATP hydrolysis to the maintenance of the bound proteins in an unfolded state. One study showing that this is the only role of cytosolic ATP in mitochondrial protein import used an experimental protocol similar to that in Figure 17-3. A precursor protein was purified and then denatured (unfolded) by urea. When added to a mixture of yeast cytosol and energized mitochondria, the denatured protein was incorporated into the matrix in the absence of cytosolic ATP. In contrast, import of the native, undenatured precursor required ATP for the normal unfolding function of cytosolic chaperones. In a second study, recombinant DNA techniques were used to construct mutant versions of certain matrix or inner-membrane proteins that were unable to fold normally into a functional conformation. These mutant proteins could be incorporated into energized mitochondria in the absence of cytosolic ATP, presumably because a chaperone was unnecessary to prevent the mutant proteins from folding.

Import of precursor proteins from the cytosol into the mitochondrial matrix always requires a proton-motive force across the inner membrane, but this electrochemical gradient is not required for binding of precursor proteins to receptors in the outer membrane. This can be demonstrated by “poisoning” mitochondria with inhibitors or uncouplers of oxidative phosphorylation such as cyanide or dinitrophenol, which dissipate the proton-motive force across the inner membrane. Although precursor proteins still can bind tightly to receptors on the poisoned mitochondria, the proteins can not be imported, either in intact cells or in cell-free systems, even in the presence of ATP and chaperone proteins.

Scientists still do not understand exactly how the membrane electric potential is used to “pull” a receptor-bound precursor protein into the matrix (see Figure 17-4, step 5). Once a protein is partially inserted into the inner membrane, it is subjected to a transmembrane potential of 200 mV (matrix space negative), which is equivalent to an electric gradient of about 400,000 V/cm. One hypothesis is that this electric potential alters the conformation of the translocation intermediate, in much the same way that a membrane electric potential affects the conformation of voltage-dependent ion channels in nerve cells. The change in protein folding could pull the precursor protein across the energized inner membrane. A related possibility is that the N-terminal matrix-targeting sequence, with its many positively charged side chains, could be “electrophoresed,” or pulled, into the matrix space by the inside-negative membrane electric potential.

ATP hydrolysis by chaperone proteins in the matrix also is required for mitochondrial protein import. These chaperones are essential for proper folding of imported proteins once they reach the matrix. In addition, the sequential binding and ATP-driven release of multiple matrix Hsc70 molecules to a precursor protein, as it emerges from the inner-membrane channel, may provide a force for pulling the unfolded protein into the matrix.

Proteins Are Targeted to Submitochondrial Compartments by Multiple Signals and Several Pathways

We turn now to targeting of proteins to submitochondrial compartments other than the matrix, namely the intermembrane space, the inner membrane, and the outer membrane (see Table 17-2). Such targeting occurs via several pathways, and for many proteins requires a second signal sequence in addition to the matrix-targeting sequence.

Intermembrane-Space Proteins

Precursors to such intermembrane-space proteins as cytochrome c1 and cytochrome b2 (subunits of the CoQH2 – cytochrome c reductase complex) carry two different N-terminal uptake-targeting sequences that get them to the intermembrane space (see Figure 17-2). The most N-terminal of the two sequences directs the N-terminus of the precursor to the matrix, where this matrix-targeting sequence is removed by the matrix protease. What happens next depends on the protein, as depicted in Figure 17-6. Some, such as cytochrome c1, follow a conservative sorting mechanism. In this case, the entire protein enters the matrix, and then the second targeting sequence directs the protein, presumably bound to matrix Hsc70, across the inner membrane to the intermembrane space. Other proteins, such as cytochrome b2, follow a nonconservative mechanism, in which the second uptake-targeting sequence functions as a stop-transfer sequence blocking translocation of the C-terminus of the precursor protein through the inner membrane. The membrane-anchored intermediate then diffuses, within the inner membrane, away from the translocation site. Finally, cleavage of the second uptake-targeting sequence by a protease in the intermembrane space releases the protein into that space. The importance of the intermembrane space–targeting sequence is evidenced by experiments with a precursor cytochrome b2 protein that lacks only this sequence. This precursor, produced by recombinant DNA techniques, accumulates in the matrix space, not in the intermembrane space.

Figure 17-6. Two pathways by which different proteins are transported from the cytosol to the mitochondrial intermembrane space.

Figure 17-6

Two pathways by which different proteins are transported from the cytosol to the mitochondrial intermembrane space. In both pathways, the precursor protein contains two N-terminal targeting sequences (top). It is delivered to Tom20/Tom22 receptors in (more...)

Unlike other mitochondrial proteins localized to the intermembrane space, cytochrome c is imported directly from the cytosol to the intermembrane space without the assistance of any targeting signals. The cytosolic form of cytochrome c, called apocytochrome c, has the same amino acid sequence as the mature protein, but lacks the covalently bound heme group found in the mature protein. Apocytochrome c, but not the mature holo form, can diffuse freely through the outer mitochondrial membrane, probably by traveling through P70. This porin-like protein (see Figure 3-35) forms channels through the phospholipid bilayer and accounts for the unusual permeability of the outer membrane to small proteins. Once apocytochrome c is in the intermembrane space of the mitochondrion, the heme group is added by the enzyme cytochrome c heme lyase. The addition of heme causes a conformational change in the protein, so that it cannot diffuse back through the outer-membrane channels, thus “locking” it into the intermembrane space.

Outer- and Inner-Membrane Proteins

Experiments with P70, a well-studied outer-membrane protein, provide clues about how proteins are targeted to the outer mitochondrial membrane. At the N-terminus of P70 there is a short matrix-targeting sequence followed by a long stretch of hydrophobic amino acids (see Figure 17-2). If the hydrophobic sequence is experimentally deleted from P70, the protein accumulates in the matrix space with its matrix-targeting sequence still attached. This finding suggests that the long hydrophobic sequence functions as a stop-transfer sequence that both prevents transfer of the protein into the matrix and anchors it as an integral protein in the outer membrane. Normally, neither the matrix-targeting nor stop-transfer sequence is cleaved from the anchored protein.

Inner-membrane proteins, in contrast, are first imported into the matrix space where the matrix-targeting sequence is removed. How these proteins then are incorporated into the inner membrane as part of multiprotein complexes is not yet known, but it probably follows the same pathway by which mtDNA-encoded inner membrane proteins are incorporated into this membrane.

The Synthesis of Mitochondrial Proteins Is Coordinated

Several proteins critical to mitochondrial function (e.g., cytochrome c oxidase and the F0F1 ATPase) are multienzyme complexes composed of some subunits synthesized in the cytosol and others synthesized in the mitochondrion. Since all of the subunits must be fabricated in appropriate ratios, the close coordination of nuclear and mitochondrial genome expression is required for the assembly of a mitochondrion. As yet, little is known about how the expression of these two genomes is coordinated in animals or plants. Studies with petite yeast strains, which lack some or all functional mtDNA (see Figure 9-43), have shown that they contain normal amounts of all nuclear-encoded mitochondrial proteins, such as cytochrome c and the F1 ATPase. Thus in yeasts, mitochondrial gene products are not essential for the expression of nuclear genes encoding mitochondrial proteins. Since mtDNA cannot be readily deleted in other organisms, it is impossible to say whether this result is true in all cells.

Yeasts and other eukaryotic microorganisms that can grow anaerobically or aerobically — in the absence or presence of O2, respectively — provide a striking example of the coordination of nuclear and mitochondrial gene expression. When grown anaerobically with glucose as a carbon source, these organisms generate ATP solely by the Embden-Meyerhof pathway (see Figures 16-3 and 16-6). When viewed under an electron microscope, anaerobically grown cells lack typical mitochondria, although they do contain some small mitochondria that have inner and outer membranes but no cristae. These mitochondria contain sufficient amounts of imported mitochondrial DNA polymerase to replicate the mtDNA normally. However, these organelles lack heme as well as cytochromes a, a3, b, c, and c1 and the F0F1 ATPase complex.

Addition of oxygen to anaerobic yeast directly activates heme synthesis. In turn, heme (not O2 itself) binds to and activates transcriptional regulatory proteins that enhance transcription of the genes for cytochrome c and many other nuclear-encoded mitochondrial proteins. Since the synthesis of heme requires oxygen, this process may be representative of a general mechanism for down-regulating expression of multiple nuclear genes that encode mitochondrial proteins during anaerobic fermentation.

Several Uptake-Targeting Sequences Direct Proteins Synthesized in the Cytosol to the Appropriate Chloroplast Compartment

The import of proteins from the cytosol to chloroplasts shares several characteristics with mitochondrial import. In both processes, imported proteins are synthesized as cytosolic precursors containing N-terminal uptake-targeting sequences that direct each protein to its correct subcompartment and are subsequently cleaved (see Table 17-1 and Figure 17-1). Protein import from the cytosol into the chloroplast stroma (equivalent to the mitochondrial matrix) occurs, as in mitochondria, at points where the outer and inner organelle membranes are in close contact. Finally, protein import into both organelles requires energy. Here, we briefly discuss targeting of proteins to the chloroplast stroma and thylakoids. We will see that despite the similarities just noted, the mechanisms of chloroplast and mitochondrial protein import differ in various ways.

Targeting to the Chloroplast Stromal Space

Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle (see Figure 16-49). The large (L) subunit of ribulose 1,5-bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes in the stromal space. The small (S) subunit of rubisco and all the other Calvin-cycle enzymes are encoded by nuclear genes and transported to chloroplasts after their synthesis in the cytosol. Rubisco, the most abundant protein in chloroplasts, is composed of eight identical L subunits, which contain the catalytic sites, and eight identical S subunits, which are necessary for full enzyme activity. This key enzyme provides an excellent example of how proteins are imported into the chloroplast stroma and assembled there into a multisubunit protein.

The S subunit of rubisco is synthesized on free cytosolic polyribosomes in a precursor form that has an N-terminal stromal-import sequence of about 44 amino acids. It is maintained in an unfolded state by binding to cytosolic chaperones. Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 17-3, have shown that they can import the S-subunit precursor after its synthesis. After the unfolded precursor enters the stromal space, it binds transiently to a stromal Hsc70 chaperone, and the N-terminal sequence is cleaved. In reactions facilitated by Hsc60 chaperonins, eight S subunits combine with the eight L subunits to yield the active rubisco enzyme.

At least three chloroplast outer-membrane proteins, including a receptor that binds the stromal-targeting sequence and a transport channel protein, and five inner-membrane proteins are known to be essential for the import process (Figure 17-7). Perhaps surprisingly, these proteins are not homologous to those of the receptor or channel proteins in the mitochondrial membrane, although their functions are analogous. The nonhomology of these chloroplast and mitochondrial proteins suggests that they may have evolved independently during evolution.

Figure 17-7. Import of rubisco small (S) subunits into the chloroplast stroma and the assembly of small and large (L) subunits into the active rubisco enzyme.

Figure 17-7

Import of rubisco small (S) subunits into the chloroplast stroma and the assembly of small and large (L) subunits into the active rubisco enzyme. Step 1: After an S-subunit precursor is fabricated in the cytosol, it presumably binds to cytosolic chaperones (more...)

The available evidence suggests that proteins are imported in the unfolded state and that import into the stroma depends on ATP hydrolysis catalyzed by stromal chaperones, whose function is similar to Hsc70 in the mitochondrial matrix. Unlike mitochondria, however, chloroplasts cannot generate an electrochemical gradient (proton-motive force) across their inner membrane. Thus protein import into the chloroplast stroma appears to be powered solely by ATP hydrolysis.

Targeting to the Thylakoids

Proteins targeted to the thylakoid membrane or lumen not only must traverse both the outer and inner chloroplast membranes to enter the stroma, but then must travel through the stroma and either be inserted into the thylakoid membrane or cross that membrane and enter the thylakoid lumen. Proteins that are destined for the thylakoid lumen, such as plastocyanin, require the successive action of two uptake-targeting sequences. The first, like that in the rubisco S subunit, targets the protein to the stroma; the second targets the protein from the stroma to the thylakoid lumen. The role of these targeting sequences has been shown in cell-free experiments measuring the uptake into chloroplasts of mutant proteins generated by recombinant DNA techniques. For instance, when the thylakoid uptake-targeting sequence is deleted from plastocyanin, the mutant protein accumulates in the stroma and is not transported into the thylakoid lumen.

Four separate thylakoid-import systems, each transporting a different set of proteins from the stroma into the thylakoid lumen, have been identified. Two of these are illustrated in Figure 17-8. The system that transports proteins such as plastocyanin employs a translocation mechanism similar to the one that translocates unfolded proteins into the endoplasmic reticulum (see later discussion). This system functions even in the absence of a pH gradient across the thylakoid membrane. Other thylakoid proteins, including a 23-kDa protein of photosystem II, are folded in the stroma and bind metal-containing redox cofactors there. In this system, the thylakoid-membrane protein Hef106 assists in translocating such folded proteins and their bound cofactors into the thylakoid lumen, and uptake is powered by the pH gradient normally maintained across the thylakoid membrane. The molecular mechanism whereby these large folded globular proteins are transported across the thylakoid membrane is currently under intense study.

Figure 17-8. Two of the four known pathways for transporting proteins from the cytosol to the thylakoid lumen.

Figure 17-8

Two of the four known pathways for transporting proteins from the cytosol to the thylakoid lumen. All such proteins are synthesized as precursors in the cytosol with two uptake-targeting sequences at their N-terminus. Shown here are the pathways for targeting (more...)

In many respects, movement of proteins from the stroma to the thylakoid lumen resembles secretion of bacterial proteins across the bacterial plasma membrane, a similarity consistent with the evolution of chloroplasts from ancestral photosynthetic bacteria. For instance, thylakoid vesicles form by inward budding from the inner membrane of protoplastids, small organelles composed only of an inner and outer membrane and a small stromal space that contains chloroplast DNA (Figure 17-9). Thus the inner proplastid membrane corresponds to the plasma membrane of the ancestral bacterium, and the chloroplast stroma corresponds to the bacterial cytoplasm. Moreover, the plasma membrane of many bacterial cells contains three protein-secreting systems that are homologs of chloroplast systems for transporting proteins from the stroma to thylakoid membranes. Secretion of most bacterial proteins occurs post-translationally and requires binding of the precursor proteins to cytosolic chaperones, much as in the post-translational uptake of unfolded proteins such as plastocyanin into the thylakoid from the stroma. Additionally, the thylakoid-targeting sequences of plastocyanin and certain other thylakoid proteins resemble the signal sequences that target bacterial proteins to cross the bacterial plasma membrane. Another bacterial system uses the pH gradient normally found across bacterial membranes to transport certain folded globular proteins from the cytosol to the cell exterior, similar to the pH-dependent uptake of folded globular proteins from the stroma into the thylakoid lumen. A final similarity occurs in the peptidases that remove certain targeting sequences. In both bacteria and chloroplasts, the sites at which these peptidases bind and cleave a target protein have a small amino acid such as glycine or alanine just N-terminal to the cleavage site and also at the position two residues toward the N-terminus.

Figure 17-9. Formation of chloroplasts from proplastids begins by the light-induced budding of the inner membrane.

Figure 17-9

Formation of chloroplasts from proplastids begins by the light-induced budding of the inner membrane. This membrane is equivalent to the plasma membrane of the ancestral photosynthetic bacterium thought to have been incorporated into an early eukaryotic (more...)


  •  Both the mitochondrion and chloroplast contain organelle DNA, which encodes organelle rRNAs and tRNAs but only a few organelle proteins.
  •  The vast majority of mitochondrial and chloroplast proteins are encoded by nuclear genes, synthesized on cytosolic ribosomes, and imported post-translationally into the organelles.
  •  Proteins destined for the mitochondrial matrix or chloroplast stroma have organelle-specific N-terminal uptake-targeting sequences that direct their entry into the organelle. After protein import, the targeting sequence is removed by proteases within the matrix or stroma.
  •  Protein import into both mitochondria and chloroplasts occurs only at sites where the inner and outer organellar membranes are in close contact.
  •  Cytosolic Hsc70 and mitochondrial import stimulation factor (MSF) are chaperones that bind the cytosolic precursors of mitochondrial proteins, keeping them in a partially unfolded form that can be translocated into mitochondria. After the unfolded proteins bind to receptors on the outer mitochondrial membrane, they are translocated through a protein-lined channel into the organelle by a process dependent on the proton-motive force across the inner membrane (see Figure 17-4). Final folding of many imported proteins is facilitated by an ATP-hydrolyzing Hsc60 chaperonin.
  •  The N-terminus of proteins imported to mitochon-drial destinations other than the matrix usually contains a second targeting sequence (see Figure 17-2). Some proteins destined for the intermembrane space or inner membrane are first imported into the matrix and then redirected; others never enter the matrix but go directly to their final location (see Figure 17-6).
  •  Import of precursor proteins into the chloropast stroma involves receptors in the outer membrane that recognize stromal-targeting sequences, channel proteins in the outer and inner membranes, and stromal chaperones (see Figure 17-7). Import requires ATP hydrolysis but not a proton-motive force. After their entry into the stroma, precursors with a second targeting sequence are redirected to the proper subcompartment, such as the thylakoid membrane or thylakoid lumen.
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