Atp

synthase subunit 9

Inner membrane (path B)

Cleavage by matrix protease

Internal sequences recognized by Oxa1

Cleavage by matrix protease

Internal sequences recognized by Oxa1

ADP/ATP antiporter

Inner membrane (path C)

Internal sequences recognized by Tom7G receptor and Tim22 complex

Cytochrome

Intermembrane space

First cleavage by Second cleavage by protease matrix protease in intermembrane space

First cleavage by Second cleavage by protease matrix protease in intermembrane space

Intermembrane space-targeting sequence

Intermembrane space-targeting sequence

Cytochrome c heme lyase

Intermembrane space

Porin (P70)

Outer membrane

Targeting sequence for the general import pore

Stop-transfer and outermembrane localization sequence used for targeting of matrix proteins (Figure 16-29, path A). A cytochrome oxidase subunit called CoxVa is a typical protein transported by this pathway. The precursor form of CoxVa, which contains an N-terminal matrix-targeting sequence recognized by the Tom20/22 import receptor, is transferred through the general import pore of the outer membrane and the inner-membrane Tim23/17 translocation complex. In addition to the matrix-targeting sequence, which is cleaved during import, CoxVa contains a hydrophobic stop-transfer sequence. As the protein passes through the Tim23/17 channel, the stop-transfer sequence blocks translocation of the C-terminus across the inner membrane. The membrane-anchored intermediate is then transferred laterally into the bilayer of the inner membrane much as type I integral membrane proteins are incorporated into the ER membrane (see Figure 16-11).

A second pathway to the inner membrane is followed by proteins (e.g., ATP synthase subunit 9) whose precursors contain both a matrix-targeting sequence and internal hydrophobic domains recognized by an inner-membrane protein termed Oxa1. This pathway is thought to involve translocation of at least a portion of the precursor into the matrix via the Tom20/22 and Tim23/17 channels. After cleavage of the matrix-targeting sequence, the protein is in-

Path A

Path B

Path C

Stop-transfer Matrix-targeting sequence sequence

Oxa1- Matrix targeting targeting mn- sequence sequence

COO-

Preprotein ■COO

Inner membrane

Mitochondrial matrix

Cleaved matrixtargeting sequences

Path B

Path C

Stop-transfer Matrix-targeting sequence sequence

Oxa1- Matrix targeting targeting mn- sequence sequence

COO-

Preprotein ■COO

Internal targeting sequences

Inner membrane

Mitochondrial matrix

Cleaved matrixtargeting sequences

Internal targeting sequences

▲ FIGURE 16-29 Three pathways for transporting proteins from the cytosol to the inner mitochondrial membrane.

Proteins with different targeting sequences are directed to the inner membrane via different pathways. In all three pathways, proteins cross the outer membrane via the Tom40 general import pore. Proteins delivered by pathways A and B contain an N-terminal matrix-targeting sequence that is recognized by the Tom20/22 import receptor in the outer membrane. Although both these pathways use the Tim23/17 inner-membrane channel, they differ in that the entire precursor protein enters the matrix and then is redirected to the inner membrane in pathway B. Matrix Hsc70 plays a role similar its role in the import of soluble matrix proteins (see Figure 16-26). Proteins delivered by pathway C contain internal sequences that are recognized by the Tom70 import receptor. A different inner-membrane translocation channel (Tim22/54) is used in this pathway. Two intermembrane proteins (Tim9 and Tim10) facilitate transfer between the outer and inner channels. See the text for discussion. [See R. E. Dalbey and A. Kuhn,

2000, Ann. Rev. Cell Devel. Biol. 16:51, and N. Pfanner and A. Geissler,

serted into the inner membrane by a process that requires interaction with Oxa1 and perhaps other inner-membrane proteins (Figure 16-29, path B). Oxa1 is related to a bacterial protein involved in inserting some inner-membrane proteins in bacteria. This relatedness suggests that Oxa1 may have descended from the translocation machinery in the endosymbiotic bacterium that eventually became the mitochondrion. However, the proteins forming the inner-

membrane channels in mitochondria are not related to the SecY protein in bacterial translocons. Oxal also participates in the inner-membrane insertion of certain proteins (e.g., subunit II of cytochrome oxidase) that are encoded by mitochondrial DNA and synthesized in the matrix by mitochondrial ribosomes.

The final pathway for insertion in the inner mitochondrial membrane is followed by multipass proteins that con tain six membrane-spanning domains, such as the ADP/ATP antiporter. These proteins, which lack the usual N-terminal matrix-targeting sequence, contain multiple internal mitochondrial targeting sequences. After the internal sequences are recognized by Tom70, a second import receptor located in the outer membrane, the imported protein passes through the outer membrane through the general import pore (Figure 16-29, path C). The protein then is transferred to a second translocation complex in the inner membrane composed of the Tim22 and Tim54 proteins. Transfer to the Tim22/54 complex depends on a multimeric complex of two small proteins, Tim9 and Tim10, that reside in the intermembrane space. These may act as chaperones to guide imported proteins from the general import pore to the Tim22/54 complex in the inner membrane. Ultimately the Tim22/54 complex is responsible for incorporating the multiple hydrophobic segments of the imported protein into the inner membrane.

Intermembrane-Space Proteins Two pathways deliver cy-tosolic proteins to the space between the inner and outer mi-tochondrial membranes. The major pathway is followed by proteins, such as cytochrome b2, whose precursors carry two different N-terminal targeting sequences, both of which ultimately are cleaved. The most N-terminal of the two sequences is a matrix-targeting sequence, which is removed by the matrix protease. The second targeting sequence is a hy-drophobic segment that blocks complete translocation of the protein across the inner membrane (Figure 16-30, path A). After the resulting membrane-embedded intermediate diffuses laterally away from the Tim23/17 translocation channel, a protease in the membrane cleaves the protein near the hydrophobic transmembrane segment, releasing the mature protein in a soluble form into the intermembrane space. Except for the second proteolytic cleavage, this pathway is similar to that of inner-membrane proteins such as CoxVa (see Figure 16-29, path A).

Cytochrome c heme lyase, the enzyme responsible for the covalent attachment of heme to cytochrome c, illustrates a second pathway for targeting to the intermembrane space. In this pathway, the imported protein is delivered directly to the intermembrane space via the general import pore without involvement of any inner-membrane translocation factors

Path A

Path B

Intermembrane spacetargeting sequence

Intermembrane spacetargeting sequence

Intermembrane spacetargeting sequence

Intermembrane spacetargeting sequence

Mitochondrial matrix

Cleaved matrix-targeting sequence

Mitochondrial matrix

Cleaved matrix-targeting sequence

▲ FIGURE 16-30 Two pathways for transporting proteins from the cytosol to the mitochondrial intermembrane space.

Pathway A, the major one for delivery to the intermembrane space, is similar to pathway A for delivery to the inner membrane (see Figure 16-29). The major difference is that the internal targeting sequence in proteins such as cytochrome b2 destined for the intermembrane space is recognized by an innermembrane protease, which cleaves the protein on the intermembrane-space side of the membrane. The released protein then folds and binds to its heme cofactor within the intermembrane space. Pathway B involves direct delivery to the intermembrane space through the Tom40 general import pore in the outer membrane. See the text for further discussion. [See R. E. Dalbey and A. Kuhn, 2000, Ann. Rev. Cell Devel. Biol. 16:51; N. Pfanner and A. Geissler, 2001, Nature Rev Mol. Cell Biol. 2:339; and K. Diekert et al., 1999, Proc. Nat'l. Acad. Sci. USA 96:11752.]

(Figure 16-30, path B). Since translocation through the Tom40 general import pore does not seem to be coupled to any energetically favorable process such as hydrolysis of ATP or GTP, the mechanism that drives unidirectional translocation through the outer membrane is unclear. One possibility is that cytochrome c heme lyase passively diffuses through the outer membrane and then is trapped within the intermembrane space by binding to another protein that is delivered to that location by one of the translocation mechanisms discussed previously.

Outer-Membrane Proteins Experiments with mitochondrial porin (P70) provide clues about how proteins are targeted to the outer mitochondrial membrane. A short matrix-targeting sequence at the N-terminus of P70 is followed by a long stretch of hydrophobic amino acids (see Figure 16-28). 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. The source of energy to drive outer membrane proteins through the general import pore has not yet been identified.

Targeting of Chloroplast Stromal Proteins Is Similar to Import of Mitochondrial Matrix Proteins

Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle, which functions in fixing carbon dioxide into carbohydrates during photosynthesis (Chapter 8). 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. The precursor forms of these stromal proteins contain an N-terminal stromal-import sequence (see Table 16-1).

Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 16-25, 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 that reside within the stromal space, eight S subunits combine with the eight L subunits to yield the active rubisco enzyme.

The general process of stromal import appears to be very similar to that for importing proteins into the mitochon-

drial matrix (see Figure 16-26). At least three chloroplast outer-membrane proteins, including a receptor that binds the stromal-import sequence and a translocation channel protein, and five inner-membrane proteins are known to be essential for directing proteins to the stroma. Although these proteins are functionally analogous to the receptor and channel proteins in the mitochondrial membrane, they are not structurally homologous. The lack of homology between these chloroplast and mitochondrial proteins suggests that they may have arisen independently during evolution.

The available evidence suggests that chloroplast stromal proteins, like mitochondrial matrix proteins, are imported in the unfolded state. Import into the stroma depends on ATP hydrolysis catalyzed by a stromal Hsc70 chaperone whose function is similar to Hsc70 in the mitochondrial matrix and BiP in the ER lumen. Unlike mitochondria, 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.

Proteins Are Targeted to Thylakoids by Mechanisms Related to Translocation Across the Bacterial Inner Membrane

In addition to the double membrane that surrounds them, chloroplasts contain a series of internal interconnected membranous sacs, the thylakoids (see Figure 8-30). Proteins localized to the thylakoid membrane or lumen carry out photosynthesis. Many of these proteins are synthesized in the cytosol as precursors containing multiple targeting sequences. For example, plastocyanin and other proteins destined for the thylakoid lumen require the successive action of two uptake-targeting sequences. The first is an N-terminal stromal-import sequence that directs the protein to the stroma by the same pathway that imports the ru-bisco S subunit. The second sequence 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, mutant plastocyanin that lacks the thylakoid-targeting sequence but contains an intact stromal-import sequence accumulates in the stroma and is not transported into the thylakoid lumen.

Four separate pathways for transporting proteins from the stroma into the thylakoid have been identified. All four pathways have been found to be closely related to analogous transport mechanisms in bacteria, illustrating the close evolutionary relationship between the stromal membrane and the bacterial inner membrane. Transport of plastocyanin and related proteins into the thylakoid lumen occurs by an SRP-dependent pathway (Figure 16-31, path A). A second pathway for transporting proteins into the thylakoid lumen

Plastocyanin COO " precursor

Thylakoid-targeting

Metal-binding precursor

Plastocyanin COO " precursor

Thylakoid-targeting

Metal-binding precursor

Tic complex

Tic complex

Plastocyanin Cleaved import sequence

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