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Protein sorting in mitochondria
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TALKINGPOINT MITOCHONDRIA are probably descended from endosymbiotic bacteria. During evolution, most of the genes of the original endosymbiont were either lost or transferred to the host nucleus. Virtually all mitochondrial proteins are therefore synthesized in the cytoplasm and must be imported into the organelle. The translocation of proteins into the mitochondrial matrix has been extensively characterized 1. Matrix proteins are usually made as larger precursors, with amino-terminal presequences that contain signals for import. The precursors are first recognized by receptors on the mitochondrial surface and are then transferred to a proteinaceous translocation complex. This complex seems to consist of two distinct translocation channels in the outer and inner membranes 2,3. Import into the matrix occurs at so-called contact sites, which are regions where the two membranes are closely apposed (Fig. 1a-d). Proteins that are targeted to the inner membrane, outer membrane and intermembrane space present more of a puzzle. However, in most cases the import of these proteins involves some or all of the same components that are used by matrix-targeted precursors. This review summarizes present knowledge on protein import to compartments other than the matrix. It concentrates primarily on targeting to the intermembrane space, since this topic has been the focus of much r e c e n t investigation and discussion. Targeting to the intermembrane space Cytochrome c reaches the intermembrane space by a relatively simple mechanism that does not use the common translocation machinery4 (Fig. lh). Apocytochrome c, which lacks the covalently attached heme, appears to insert directly into the lipid bilayer of the outer membrane. The driving force for import is provided by cytochrome c heme lyase (CCHL), which catalysesthe attachment of heme to apocytochrome c in the intermembrane space 5-7. It has been suggested that apocytochrome c binds to CCHL after inserting into the B. S. Glick, E. M. Beasley and G. Schatz are
at the Biozentrumder Universitfit Basel, Abt. Biochemie, CH-4056Basel, Switzerland. © 1992,ElsevierSciencePublishers, (UK)
Most polypeptides that are imported into the mitochondrial matrix use a common translocation machinery. By contrast, proteins of the other mitochondrial compartments are imported by a variety of different mechanisms. Some of these proteins completely bypass the common translocation machinery, others use only the outer membrane components of this machinery, and still others use components of this machinery from both the outer and inner membranes. Import to the intermembrane space compartment provides examples of all three possibilities.
outer membrane, and that heme attachment then causes complete translocation across this membrane 6. Alternatively, the interaction with CCHL may occur after apocytochrome c is already soluble in the intermembrane space 7,8. In any case, heme attachment is necessary to retain cytochrome c inside the mitochondria 5-8. This pathway for cytochrome c localization is found only in eukaryotes; the analogous protein in bacteria is synthesized inside the cell and exported across the plasma membrane. The import of cytochrome c into mitochondria has been termed 'nonconservative sorting', because the sorting mechanism has not been conserved during evolution 9. Like cytochrome c, CCHL is synthesized as the mature-length protein ~°. However, by contrast to cytochrome c, CCHL is imported through the same outer membrane channel that is used by matrix-targeted precursors TM. The mature protein appears to be bound to the outer surface of the inner membrane 7 (Fig. lg). Import of CCHL probably does not involve the common translocation machinery in the inner membrane, and the force that pulis this precursor across the outer membrane is unknown. As this second pathway to the intermembrane space has no counterpart in prokaryotes, the sorting of CCHL is also nonconservative 1°. A third targeting mechanism is used by a group of intermembrane-space enzymes that includes cytochrome b 2 (Fig. l f ) and cytochrome ci (Fig. le), and probably also cytochrome c perox-
idase and mitochondrial creatine kinase 1. These proteins are synthesized with transient presequences. The aminoterminal portion of each of these presequences resembles a matrix-targeting signal; the carboxy-terminal portion contains a hydrophobic stretch, and functions as the intermembrane space targeting domain (Fig. 2). Two models have been proposed to explain how this targeting information is decoded by the mitochondria: In both models, the precursor interacts with the common translocation machinery in the outer and inner membranes. According to the 'stop-transfer' hypothesis, the aminoterminal part of the presequence is imported into the matrix, but the intermembrane space targeting domain arrests further translocation through the inner membrane u-I3 (Fig. 3). The import channels in the two membranes would then dissociate 2, allowing the mature portion of the protein to cross the outer membrane to the intermembrane space. Such a sorting pathway would be nonconservative. An alternative possibility is that these proteins are first imported completely into the matrix, and are then translocated back across the inner membrane to the intermembrane space 9,14 (Fig. 3). The intermembrane space targeting domains would thus function as signals for reexport from the matrix. This model is termed 'conservative sorting', because the second part of the pathway is thought to involve a n evolutionarily conserved bacterial-type export machinery 9.
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sections summarize the evidence that supports this conclusion. In previous discussions of the two models, proteins that use different sorting mechanisms were sometimes placed in the same category. To help clarify this issue, we will first define some key terms for describing protein sorting in mitochondria.
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Figure 1 Sorting pathways of imported mitochondrial proteins. Proteins are grouped according to the compartment to which they are initially targeted. Unless otherwise stated, the diagram refers to yeast mitochondria and omits post-translational modifications such as proteolytic cleavage and the formation of homooligomers. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. (See text and Ref. i for further details.) Matrix: (a) Subunit 9 of the Fo portion of the mitochondrial ATPase. In Neurospora crassa and higher eukaryotes, this precursor is apparently imported into the matrix, where the presequence is cleaved off 27. The mature protein then integrates into the lipid bilayer of the inner membrane. (b) The #subunit of the F1-ATPase. (c) The mitochondrial isoenzyme alcohol dehydrogenase II1. (d) The iron-sulfur protein of complex III of the respiratory chain. Intermembrane space: (e) Cytochrome c1 is initially attached to the inner membrane by its transient presequence (shown in outline). Our results indicate that this precursor follows a stop-transfer pathway (,). (f) Cytochrome b2. (g) Cytochrome c heine lyase. Although import is shown as occurring at contact sites, it is possible that this protein can be imported through unattached outer membrane regions. (h) Cytochrome c. It is not known whether import of this protein takes place in unattached outer membrane regions, as shown, or at contact sites. At physiological salt concentrations, cytochrome c is bound to cytochrome oxidase. Inner membrane: (i) Subunit Va of cytochrome oxidase. (j) The ADP/ATP translocator. Outer membrane: (k) The MAS70 protein. (I) Mitochondrial porin.
Why is it important to distinguish between these two models~ If the conservative sorting hypothesis is correct, then the impressive advances in the field of bacterial protein export 15can be applied to studies of mitochondrial biogenesis. On the other hand, if certain sequences can act as stop-transfer sig-
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nals in mitochondria, an understanding of this process would shed light on the nature of mitochondrial import sites 2 and possibly on the mechanisms of translocation arrest in other systems. Our investigations of c y t o c h r o m e s b 2 and c~ suggest that these proteins foJ!ow a stop-transfer pathway. The following
Targeting followed by assembly In the past, proteins have been assigned to the Various mitochondrial compartments ~in a somewhat arbitrary and inconsistent fashion. The reason is that the proteins of a given compartment may be imported by several different routes. For example, cytochrome cl is generally considered to be targeted to the intermembrane space TM and subunit IV of cytochrome oxidase (CoxlV) is regarded as a matrix-targeted precursor 17, yet both proteins end up tightly associated with the inner membrane. We propose that for studies of mitochondrial biogenesis, proteins should be classified according to the compartment to which they are initially targeted. The subsequent assembly of oligomeric complexes should be considered separately. A targeting mechanism may be used by _an entire class of precursors, whereas each protein has a unique assembly pathway. Intramitochondrial sorting is therefore a two-stage process, with different signals and catalysts required for each stage. In the example given above, cytochrome g is targeted to the intermembrane space and CoxIV is targeted to the matrix, and both of these proteins then assemble into complexes in the inner membrane. To apply this classification system, we suggest the following definitions: (1) A protein is targeted to the matrix if every residue of the mature polypeptide is exposed to the matrix at some time during import. (2) A protein is targeted to the intermembrane space if every residue of the mature polypeptide is exposed to the intermembrane space at some time during import. (3) A protein is targeted to the inner membrane if part of the mature polypeptide is translocated across the inner membrane at contact sites, and part of the mature polypeptide never crosses the inner membrane. (4) A protein is targeted to the outer membrane if part but not all of the polypeptide crosses the outer membrane.
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The presequences of cytochromes b 2 and cI contain bipartite targeting signals. (a) The general structure of a presequence with intermembrane space targeting information. The amino-terminal portion of the presequence resembles a matrix-targeting signal; the carboxy-terminal portion contains a hydrophobic stretch (represented by the black box) that is necessary for targeting to the intermembrane space. The presequence is cleaved twice, first by the soluble matrix processing protease (~) and then by a protease facing the intermembrane space (A). (b)Yeast cytochrome b2; (c) yeast cytechrome cl; both cytochromes conform to the scheme outlined in (a). The hydrophobic stretch in each presequence is underlined, and charged amino acids are indicated. The first cleavage site for cytochrome cI has not been identified, but it is probably within the region marked by the bracket. For the amino acids shown in bold, mutations have been isolated that weaken or inactivate the intermembrane space targeting signals (Ref. 29; E. Beasiey, unpublished). In the cytochrome b2 presequence, this targeting signal has been shown to include residues outside of the hydrophobic stretch. (d) The presequence of the iron-sulfur protein is also cleaved in two steps, first by the general matrix processing protease (A) and then by a different enzyme (A). Both processing events probably occur in the matrix25. It is thus unlikely that this presequence contains intermembrane space targeting information.
This simplified scheme assumes that ample, mouse dihydrofolate reductase during translocation through both mem- (DHFR) can be targeted to the matrix by branes at contact sites, the precursor the presequence of subunit 9 of the Fo polypeptide chain is shielded from the complex 19 (Fig. la), to the intermemintermembrane space. This assumption brane space by the presequence of may not always be correct 2,3J8. For pro- cytochrome cl TM (Fig. le), to the inner teins such as cytochromes b2 and c~, membrane by internal targeting sethe distinction between targeting to the quences in the ADP/ATP translocator 2° matrix and to the intermembrane space (Fig. lj), or to the outer membrane by is unambiguous only for a stop-transfer the amino-terminal portion of the mechanism; if these proteins followed MAS70 protein 21(Fig. lk). a re-export pathway, this set of definitions would have to be modified. Fig. The iron-sulfur protein of complex III is 1 illustrates the import pathways of sev- targeted to the matrix eral mitochondrial proteins, grouped acThe definitions given above are useful cording to the definitions given above. for distinguishing between proteins This classification system is con- that reach the same intramitochondrial sistent with the results of gene-fusion location by different mechanisms. For experiments, in which a precursor's tar- example, in complex III of the respiratory geting signal is tested for its ability to chain, cytochrome ci and the iron-sulfur direct a passenger protein to a specific protein both have their active sites at mitochondria] compartment. For ex- the external face of the inner mem-
brane 22. T h e iron-sulfur protein is first translocated completely into the matrix, and then inserts into complex IlI by a process that probably resembles the corresponding bacterial assembly reaction 9,12,23,24(Fig. ld). Thus for the ironsulfur protein the conservative sorting model is clearly appropriate. However, there is disagreement about whether this model can be extended to cytochrome 619'12-14 . At first glance, cytochrome cl and the iron-sulfur protein might be expected to use similar sorting mechanisms. The presequences of both proteins are cleaved in two steps upon import 16,24. However, only the cytochrome cl presequence contains a bipartite targeting signal (Fig. 2). The amino-terminal portion of the cytochrome cl presequence is cleaved off by the matrix protease, and the intermembrane space targeting
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import of cytochrome b 2 into isolated mitochondria, a large percentage of the (a) S t o p - t r a n s f e r (b) C o n s e r v a t i v e s o r t i n g molecules were apparently present in the matrix TM. Second, when the matrixlocalized hsp60 chaperone was inactivated in a OM ......... : , OM temperature-sensitive yeast mutant, the intermediatesized form of cytochrome b 2 accumulated in the cells, suggesting that hsp60 function was required for reexporting cytochrome b 2 to the intermembrane space 23. We decided to re-examine these findings, since insights into the dynamic nature of the mitochondrial import machinery had revived our interest in IM IM the stop-transfer model 12. To analyse the topology of imported cytochrome b2, Figure 3 we selectively ruptured the Two models for the targeting of cytochromes b2 and c1 to the intermembrane space. The wavy line with mitochondrial outer mem'+' signs represents the amino-terminal part of the presequence, and the solid rectangle represents brane by osmotic shock, the intermembrane space targeting domain. OM, outer membrane; IM, inner membrane. (a) Stop transand then added protease fer. The intermembrane space targeting domain arrests translocation through the inner membrane. The to digest molecules that amino-terminal portion of the presequence is then cleaved off by the matrix protease. The mature domain of the precursor continues to cross the outer membrane to the intermembrane space, where the were outside the inner remainder of the presequence is removed by a second protease. The hsp60 chaperone should not be membrane. This method involved in this pathway; matrix ATP may or may not be required. (b) Conservative sorting. The precurdid not reveal any matrixsor is imported completely into the matrix, where it interacts with hsp60. The intermembrane space tarlocalized sorting intergeting domain is then recognized by a bacterial-type export machinery, which translocates the protein mediates 13. By contrast, in back across the inner membrane to the intermembrane space. Matrix ATP shoutd be required both for previous studies the mitothe initial import into the matrix and for release from hsp60. Two-step cleavage would occur as in the stop-transfer model. chondria were subfractionated with the detergent digitonin; after this treatdomain is removed by a second pro- act as a signal-anchor sequence, since the ment the imported intermediate form of tease at the outer face of the inner protein apparently does not integrate cytochrome b 2 behaved like a soluble membrane u,~6. By contrast, the pre- into the lipid bilayer of the inner mem- matrix protein ~4. Although we could sequence of the iron-sulfur protein brane 24. Moreover, there is no evidence reproduce this result, additional experseems to contain only matrix-targeting that this hydrophobic stretch acts as a iments indicated that the cytochrome b2 information (Fig. 2). The iron-sulfur sorting signal. For these reasons we intermediate was not actually located in protein belongs to a class of precursors suggest that it is misleading to describe the matrix~, but was bound to the inner whose presequences are cleaved twice the assembly of the iron-sulfur protein membrane and facing the intermemin the matrix by two different en- as 're-export'. Similar arguments apply brane space ~3. A likely explanation for zymes25; other examples include the to other polypeptides that assemble the aberrant behavior of this protein soluble matrix proteins ornithine trans- into the inner membrane after being im- during digitonin fractionation is that the cytochrome b 2 intermediate was not carbamylase and malate dehydrogenase. ported into the matrix 17,27. integrated into the lipid bilayer, and This two-step cleavage probably functherefore was solubilized at lower detions to ensure correct processing Cytochromes b2 and c~: stop.transfer or tergent concentrations than endogenous rather than to specify intramitochon- conservative sorting? drial sorting 26. Initial results from our laboratory were inner membrane proteins 13. It is still unresolved whether or not Thus unlike cytochrome cl, which is consistent with a stop-transfer mechan intermembrane space-targeted pre- anism for the targeting of cytochromes hsp60 is necessary for targeting to the cursor, the iron-sulfur protein is tar- b 2 and c1: during import of a precursor intermembrane space. Using the same geted to the matrix. This p~rotein then containing the cytochrome ci pre- temperature-sensitive mutant as previassembles into complex II[ by an sequence, no intermediate forms were ously, we were unable to confirm that unknown mechanism. Although the found inside the inner membrane ~2. inactivation of hsp60 prevents the matumature iron-sulfur protein contains a However, other groups subsequently ration of cytochrome b2. In our hands, hydrophobic stretch near its amino ter- reported two results that s u p p o r t e d t h e cytochromes b2 and ci were processed minus 22, this domain probably does not conservative sorting model. First, after normally when hsp60 was inactivated 13.
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Moreover, cytochrome b 2 maturation was still observed after hsp60 had been depleted from yeast cells with the aid of a regulatable promoter (R. L. Hallberg, pers. commun.). Recent in vitro experiments provide a further test of hsp60 function. A fusion protein was constructed in which a large portion of the cytochrome b 2 precursor was joined to the amino terminus of DHFR28. During import, methotrexate was added to prevent passage of the DHFR domain across the outer membrane. Despite the methotrexate block, both cleavage reactions of the presequence took place, indicating that part of the precursor was exposed to the second processing protease in the intermembrane space. The conclusion from the conservative sorting model was that re-export could begin even though import across the outer membrane was incomplete; thus a species was formed that spanned three membranes. In support of this interpretation, the mature fusion protein was associated with hsp60 after the mitochondria were disrupted with digitonin 28. However, it was not rigorously excluded that binding to hsp60 took place after the digitonin treatment. According to the stop-transfer model, this fusion protein was arrested with its amino terminus in the matrix, its DHFR domain outside the outer membrane, and its cytochrome b 2 moiety facing the intermembrane space. Cleavage by the second processing protease would therefore generate a mature protein that spanned only the outer membrane. Such a species would be on a 'deadend' pathway, unable to finish translocation to the intermembrane space. Indeed, our preliminary results indicate that this protein cannot cross the outer membrane even when the methotrexate is removed 03. Glick, unpublished). Additional work is needed to distinguish between the different proposed topologies of this intermediate. The two models for intermembrane space targeting were also tested by the following approaches: (1) Since matrixlocalized ATP is essential for complete import across the inner membrane ~8 and for the release of proteins from hsp60 ~9, the conservative sorting model predicts that matrix ATP should be required for targeting to the intermembrane space (Fig. 3). In fact, fusion proteins containing the presequences of either cytochrome cl or cytochrome b 2 were imported to the intermembrane space and processed normally in ATP-
depleted mitochondria 13. With authentic cytochrome c~, depletion of matrix ATP had no effect either on import or on the complex maturation pathway of this protein 29. (2) According to the stoptransfer model, the intermembrane :space targeting domain arrests translocation at an early stage in the import reaction. In the conservative sorting model this targeting domain acts later in the pathway, after the precursor has been transported into the matrix. A kinetic experiment showed that translocation was arrested at an early step, as expected for a stop-transfer mechanism 03. Glick and K. Cunningham, unpublished). (3) It is possible that; depending upon the conditions, some proteins can use either a stop-transfer or a conservative sorting mechanism. To test this idea, we artificially introduced a polypeptide containing the cytochrome cl presequence into the matrix. This protein was not retranslocated to the intermembrane space, suggesting that mitochondria lack a signal sequencedependent export system for proteins imported from the cytoplasm 03. Glick and K. Cunningham, unpublished). While none of these experiments is conclusive by itself, we believe that the combined data strongly favor the stoptransfer hypothesis. However, a consensus on this issue is still lacking. Table I summarizes the experimental evidence for each of the two models, together with alternative interpretations. Since we have criticized the conservative sorting model, we should also discuss some weaknesses of the evidence for a stop-transfer mechanism. The apparent lack of an hsp60 requirement for targeting to the intermembrane space could simply mean that cytochromes b2 and c~ do not need to interact with this chaperone as they pass through the matrix. Moreover, our inability to detect matrix-localized intermediates may indicate that reexport is faster than import. If re-export can begin before import into the matrix is complete 28, then under normal conditions one might not observe intermediates that were entirely inside the inner membrane. It is even possible that import and re-export are obligatorily coupled, so that the protein we artificially introduced into the matrix would not have been a productive sorting intermediate. The simultaneous import-reexport hypothesis implies that an intermembrane space targeting domain can bind to hsp60 during import28; such an interaction could account for the trans-
location arrest observed in our kinetic study. These arguments do not explain why cytochrome ci still undergoes normal targeting and maturation in ATPdepleted mitochondria 29. However, it is conceivable that import into such mitochondria occurs by a non-physiological mechanism, since even matrix-targeted precursors accumulate between the two membranes when ATP levels are low TM.
Intermembrane space targeting signals One indirect argument against the stop-transfer model has been that several proteins with extended hydrophobic stretches are imported through both membranes into the matrix24,27. Our analysis of intermembrane space targeting signals provides a solution to this problem. A genetic study of the presequence of cytochrome b 2 (Fig. 2) confirmed that the hydrophobic stretch is important for targeting to the intermembrane space (E. Beasley, unpublished). Unexpectedly, the introduction of Pro residues into this stretch severely disrupted the targeting signal; for example, converting Leu62 to Pro inhibited targeting more effectively than placing Arg at the same position. Thus the function of this domain apparently depends both on its hydrophobicity and on its conformation. Efficient targeting of cytochrome b 2 to the intermembrane space also requires basic residues upstream of the hydrophobic stretch, and Glu at position +1 of the mature protein (Fig. 2). These data support the suggestion that intermembrane space targeting domains resemble bacterial signal sequences 9. Indeed, the second cleavage of the cytochrome b 2 presequence is catalysed by an enzyme that is related to bacterial leader peptidase 3°. However, such observations do not imply that sorting occurs in the same way in bacteria and in mitochondria. We suggest instead that prokaryotic-type targeting sequences have been adopted during evolution as signals for a uniquely eukaryotic stop-transfer pathway. Because these stop-transfer signals are relatively complex, a hydrophobic sequence can be present in a precursor without necessarily arresting translocation through the inner membrane.
Sorting to the outer and inner membranes Proteins are sorted to the outer membrane by many different mechanisms. The insertion of mitochondrial porin (Fig. 11) seems to require the common translocation machinery in the 457
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Table I. Stop-transfer or conservative sorting? Evidence for stop-transfer
Criticisms
Evidence for conservative sorting
Criticisms
(1) No sorting intermediates were found in the matrix for either cytochrome b 2 or cytochrome
Re-export might be very rapid. Under some conditions, reexport may be simultaneous with import28.
(1) The intermediate form of cytochrome b 2 co-fractionated with soluble matrix proteins when mitochondria were treated with digitonin14.
(2) The matrix-localized hsp60 chaperone was not required for the maturation of cytochromes b2 and c1 in vivo 13.
These results do not match a previous observation, which suggested that hsp60 was necessary for cytochrome b2 maturation in vivo 23, Moreover, re-export might still be possible in the absence of hsp60.
The digitonin method seems to give misleading results, because the cytochrome b 2 intermediate is not integrated into the lipid bilayer of the inner membrane13,
(2) When hsp60 was inactivated in a temperature-sensitive yeast mutant, the intermediate form of cytochrome b2 was detected in the cells 23.
This in vivo effect was not observed in another study13. Moreover, inactivation of hsp60 did not prevent cytochrome b 2 maturation in vitro 23,
(3) When import into the matrix was prevented by depleting the mitochondria of ATP, several intermembrane space-targeted proteins were still imported and processed normally13'29,
This phenomenon could represent a non-physiological side pathway, since even matrixtargeted precursors reach the intermembrane Space in ATPdepleted mitochondria18.
(4) When an intermembrane space-targeted precursor was placed in the matrix by artificial means, it was not re-exported a.
This precursor reached the matrix . by an abnormal pathway, and thus may have been unable to interact with the re-export machinery.
(3) A protein containing the cytochrome b 2 presequence could be trapped with part of the protein outside the mitochondria and another part in the intermembrane space. After detergent solubilization, this protein was bound to hsp6028.
Association with hsp60 might have occurred after the detergent treatment. In addition, this trapped protein apparently is not a productive intermediate on the pathway of import to the intermembrane spaceb.
(4) Some proteins with extended hydrophobic stretches are imported into the matrix24'27.
(5) A functional intermembrane space-targeting domain in a precursor protein caused a translocation arrest at an early stage of importa.
If hsp60 bound to this targeting domain during import28, translocation might have been slowed down.
A hydrophobic stretch by itself does not necessarily function as an intermembrane space targeting signal. These signals consist of several domainsc.
(5) The iron-sulfur protein of complex III is imported completely into the matrix before assembling into the inner membrane12,24.
The iron-sulfur protein probably follows a different sorting pathway than cytochromes b 2 and c1. Unlike these two cytechromes, the iron-sulfur protein does not have an intermembrane space targeting domain in its presequence25.
Ci12,13,29.
aB. Glick and K. Cunningham, unpublished; bB. Glick, unpublished; °E. Beasley, unpublished.
outer membrane 31. The import receptor MOM19 associates with the outer membrane by interacting with other components of the translocation complex32; thus for this protein the targeting and assembly reactions may be identical. A second import receptor, called MAS70 in yeast, is targeted to the outer membrane by its amino-terminal domain, which includes an extended hydrophobic stretch that functions as a membrane anchor 33 (Fig. lk). In experiments with chimeric precursors containing the MAS70 hydrophobic stretch, it was observed that insertion into the outer membrane could occur in either orientation, depending upon the sequence located upstream of the membrane anchor 21. MOM72, the Neurospora homologue of MAS70, requires MOM19 for its insertion34. Perhaps the nkost unusual mechanism for outer membrane localization is that of monoamine o~xidase B: this protein contains a targeting signal near its carboxyl terminus 35, and membrane insertion in vitro requires ubiquitin 36.
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As discussed earlier, a number of proteins are initially targeted to the matrix or intermembrane space before assembling into oligomeric complexes in the inner membrane. Proteins can also be targeted directly to this membrane. For example, the ADP/ATP translocator apparently moves from the inner membrane import channel into the lipid bilayer without passing through the matrix (Fig. l j); i.e. this protein follows a nonconservative sorting pathway9,27. Subunit Va of cytochrome oxidase (CoxVa; Fig. li) is synthesized with a transient presequence, but targeting to the inner membrane also requires a hydrophobic stretch near the carboxyl terminus of the mature protein 37. Although the import mechanism of CoxVa is not known in detail, this hydrophobic sequence might act as an internal stop-transfer signal. Both the ADP/ATP translocator and CoxVa follow at least part of the same pathway that is used by matrix-targeted precursors 31,37. An entirely different import mechanism may exist for subunit VI of
complex III, as this acidic protein lacks a typical matrix-targeting signal38. Conclusion
Eukaryotic ceils have developed a generalized system for transporting proteins from the cytoplasm into the mitochondrial matrix. Many proteins reach the other mitochondrial compartments by diverging from this matrix import pathway at different stages. Mitochondrial protein sorting thus seems to be an example of evolutionary opportunism; with individual proteins selectively using components of a common translocation machinery for their own unique sorting mechanisms. References 1 Glick, B. and Schatz, G. (1991) Annu. Rev. Genet. 25, 21-44 2 Glick, B., Wachter, C. and Schatz, G. (1991) Trends Cell Biol. 1, 99-103 3 Planner, N., Rassow, J., van der Klei, I. and Neupert, W. (1992) Cell 68, 999-1002 4 Stuart, R. A. and Neupert, W. (1990) Biochimie 72, 115-121 5 Dumont, M. E., Ernst, J. F. and Sherman, F. (1988) J. Biol. Chem. 263, 15928-15937
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6 Nicholson, D. W., Hergersberg, C. and Neupert, W. (1988) J. Biol. Chem. 263, 19034-19042 7 Dumont, M. E., Cardillo, T. S., Hayes, M. K. and Sherman, F. (1991) Mol. Cell. Biol. 11, 5487-5496 8 Hakvoort, T. B. M., Sprinkle, J. R. and Margoliash, E. (1990) Proc. Natl Acad. Sci. USA 87, 4996-5000 9 Hartl, F-U. and Neupert, W. (1990) Science 247, 930-938 10 Lill, R. et al. (1992) EMBO J. 11, 449-456 11 Hurt, E. C. and van Loon, A. P. G. M. (1986) Trends Biochem. Sci. 11, 204-206 12 van Loon, A. P. G. M. and Schatz, G. (1987) EMBO J. 6, 2441-2448 13 Glick, B. S. et al. (1992) Cell 69, 809-822 14 Hartl, F-U., Ostermann, J., Guiard, B. and Neupert, W. (1987) Cell 51, 1027-1037 15 Wickner, W., Driessen, A. J. M. and Hartl, F-U. (1991) Annu. Rev. Biochem. 60, 101-124 16 van Loon, A. P. G. M., Br~ndli, A. W. and
IMPRESSIVE PROGRESS towards an understanding of the forces that stabilize the folded structures of globular proteins has been described in recent reviews in T I B S ~ and elsewhere 2,3. For most of the past 30 years it has been commonly supposed that the hydrophobic interaction makes a prominent contribution to the stability of the native form, as proposed by Kauzmann4. However, it has recently repeatedly been claimed that the contrary is true ~ . That no consensus now exists is demonstrated by the fact that two highly respected reviewers have examined the same body of data and have drawn opposite conclusions 2,3. This review summarizes the way in Which these divergent opinions arose and scrutinizes some of the arguments for reversing Kauzmann's hypothesis, especially those based on observations of relative stabilities of proteins of differing hydrophobicity. After some deliberation, it then appears that all available data are quite consistent with the conclusion that structural reorganization of water adjacent to non-polar groups opposes unfolding, as originally proposed.
Protein unfolding and dissolution of liquid .... hydrocarbons in water Protein denaturation resembles the formation of aqueous solutions of simple hydrocarbons in that both involve N. Muller is at the Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
© 1992,ElsevierSciencePublishers, (UK)
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27 Mahlke, K. et al. (1990) Eur. J. Biochem. 192,
551-555 28 Koll, H. et al. (1992) Ceil 68, 1163-1175 29 Wachter, C., Schatz, G. and Glick, B.S. EMBO J.
(in press) 30 Behrens, M., Michaelis, G. and Pratje, E. (1991) Mol. Gen. Genet. 228, 167-176 31 Pfaller, R. et al. (1988) J. Cell Biol. 107,
2483-2490 32 Schneider, H. et al. (1991) Science 254,
1659-1662 33 Hase, T., MOiler, U., Riezman, H. and Schatz, G. (1984) EMBO J. 3, 3157-3164 34 S6llner, T. et al. (1990) Cell 62,107-115 35 Mitoma, J. and Ito, A. (1992) J. Biochem. 111,
20-24 36 Zhuang, Z. and McCauley, R. (1989) J. Biol. Chem. 264, 14594-14597 37 Miller, B. R. and Cumsky, M. G. (1991) J. Cell Biol. 112, 833-841 38 van Loon, A. P. G. M. et al. (1984) EMBO J. 3,
1039-1043
Does hydrophobichydration destabilize protein native structures?
Water in the immediate vicinity of a non-polar solute has characteristically low entropy and high heat capacity at 25°C. Common opinion has been that the insolubility of such species is caused by thermodynamic changes associated with the formation of these layers of abnormal water, 'hydrophobic hydration'. Recently, however, it has been proposed instead that hydrophobic hydration favors solution of hydrocarbons, or hydrocarbon sidechains, in water and therefore promotes protein unfolding. It is argued here that available data do not convincingly support this hypothesis.
transfer of non-polar groups from a nonaqueous to an aqueous environment4,5,7,9. This is characteristically accompanied by a large increase in the heat capacitY, proportional to the surface area of the hydrocarbon solute 1° or, for a protein, to the non-polar area exposed to water upon unfolding u. I t is generally interpreted as a manifestation of special structural or thermodynamic properties of water that is in contact with a non-polar group. Formation of this special kind of water is called hydrophobic hydration, and the resulting thermodynamic changes, here labelled with the subscript hph, constitute the hydrophobic effect. Observation of hydrocarbon solutions provides an opportunity to study this effect with minimal
interference from other interactions. Transfer of an alkane molecule from the pure liquid (1) to a water solution (w) is opposed by a large increase in the unitary Gibbs energy4, AG0(I-+w)
=
AH0(1-+w)
= - RTlnX,
-
TAS0(1--+w) (1)
where X is the mole fraction in the saturated solution, AH° is the enthalpy change and AS° is the entropy change. Near room temperature, the dominant term is TAS° (1-+w), which is large and negative. It has been reported that for an assortment of hydrocarbons the transfer enthalpies and entropies at any (Kelvin) temperature are well represented by the following empirical equations9:
459
TALKINGPOINT MITOCHONDRIA are probably descended from endosymbiotic bacteria. During evolution, most of the genes of the original endosymbiont were either lost or transferred to the host nucleus. Virtually all mitochondrial proteins are therefore synthesized in the cytoplasm and must be imported into the organelle. The translocation of proteins into the mitochondrial matrix has been extensively characterized 1. Matrix proteins are usually made as larger precursors, with amino-terminal presequences that contain signals for import. The precursors are first recognized by receptors on the mitochondrial surface and are then transferred to a proteinaceous translocation complex. This complex seems to consist of two distinct translocation channels in the outer and inner membranes 2,3. Import into the matrix occurs at so-called contact sites, which are regions where the two membranes are closely apposed (Fig. 1a-d). Proteins that are targeted to the inner membrane, outer membrane and intermembrane space present more of a puzzle. However, in most cases the import of these proteins involves some or all of the same components that are used by matrix-targeted precursors. This review summarizes present knowledge on protein import to compartments other than the matrix. It concentrates primarily on targeting to the intermembrane space, since this topic has been the focus of much r e c e n t investigation and discussion. Targeting to the intermembrane space Cytochrome c reaches the intermembrane space by a relatively simple mechanism that does not use the common translocation machinery4 (Fig. lh). Apocytochrome c, which lacks the covalently attached heme, appears to insert directly into the lipid bilayer of the outer membrane. The driving force for import is provided by cytochrome c heme lyase (CCHL), which catalysesthe attachment of heme to apocytochrome c in the intermembrane space 5-7. It has been suggested that apocytochrome c binds to CCHL after inserting into the B. S. Glick, E. M. Beasley and G. Schatz are
at the Biozentrumder Universitfit Basel, Abt. Biochemie, CH-4056Basel, Switzerland. © 1992,ElsevierSciencePublishers, (UK)
Most polypeptides that are imported into the mitochondrial matrix use a common translocation machinery. By contrast, proteins of the other mitochondrial compartments are imported by a variety of different mechanisms. Some of these proteins completely bypass the common translocation machinery, others use only the outer membrane components of this machinery, and still others use components of this machinery from both the outer and inner membranes. Import to the intermembrane space compartment provides examples of all three possibilities.
outer membrane, and that heme attachment then causes complete translocation across this membrane 6. Alternatively, the interaction with CCHL may occur after apocytochrome c is already soluble in the intermembrane space 7,8. In any case, heme attachment is necessary to retain cytochrome c inside the mitochondria 5-8. This pathway for cytochrome c localization is found only in eukaryotes; the analogous protein in bacteria is synthesized inside the cell and exported across the plasma membrane. The import of cytochrome c into mitochondria has been termed 'nonconservative sorting', because the sorting mechanism has not been conserved during evolution 9. Like cytochrome c, CCHL is synthesized as the mature-length protein ~°. However, by contrast to cytochrome c, CCHL is imported through the same outer membrane channel that is used by matrix-targeted precursors TM. The mature protein appears to be bound to the outer surface of the inner membrane 7 (Fig. lg). Import of CCHL probably does not involve the common translocation machinery in the inner membrane, and the force that pulis this precursor across the outer membrane is unknown. As this second pathway to the intermembrane space has no counterpart in prokaryotes, the sorting of CCHL is also nonconservative 1°. A third targeting mechanism is used by a group of intermembrane-space enzymes that includes cytochrome b 2 (Fig. l f ) and cytochrome ci (Fig. le), and probably also cytochrome c perox-
idase and mitochondrial creatine kinase 1. These proteins are synthesized with transient presequences. The aminoterminal portion of each of these presequences resembles a matrix-targeting signal; the carboxy-terminal portion contains a hydrophobic stretch, and functions as the intermembrane space targeting domain (Fig. 2). Two models have been proposed to explain how this targeting information is decoded by the mitochondria: In both models, the precursor interacts with the common translocation machinery in the outer and inner membranes. According to the 'stop-transfer' hypothesis, the aminoterminal part of the presequence is imported into the matrix, but the intermembrane space targeting domain arrests further translocation through the inner membrane u-I3 (Fig. 3). The import channels in the two membranes would then dissociate 2, allowing the mature portion of the protein to cross the outer membrane to the intermembrane space. Such a sorting pathway would be nonconservative. An alternative possibility is that these proteins are first imported completely into the matrix, and are then translocated back across the inner membrane to the intermembrane space 9,14 (Fig. 3). The intermembrane space targeting domains would thus function as signals for reexport from the matrix. This model is termed 'conservative sorting', because the second part of the pathway is thought to involve a n evolutionarily conserved bacterial-type export machinery 9.
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sections summarize the evidence that supports this conclusion. In previous discussions of the two models, proteins that use different sorting mechanisms were sometimes placed in the same category. To help clarify this issue, we will first define some key terms for describing protein sorting in mitochondria.
M (c)
(b)
X
S
o F
Figure 1 Sorting pathways of imported mitochondrial proteins. Proteins are grouped according to the compartment to which they are initially targeted. Unless otherwise stated, the diagram refers to yeast mitochondria and omits post-translational modifications such as proteolytic cleavage and the formation of homooligomers. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. (See text and Ref. i for further details.) Matrix: (a) Subunit 9 of the Fo portion of the mitochondrial ATPase. In Neurospora crassa and higher eukaryotes, this precursor is apparently imported into the matrix, where the presequence is cleaved off 27. The mature protein then integrates into the lipid bilayer of the inner membrane. (b) The #subunit of the F1-ATPase. (c) The mitochondrial isoenzyme alcohol dehydrogenase II1. (d) The iron-sulfur protein of complex III of the respiratory chain. Intermembrane space: (e) Cytochrome c1 is initially attached to the inner membrane by its transient presequence (shown in outline). Our results indicate that this precursor follows a stop-transfer pathway (,). (f) Cytochrome b2. (g) Cytochrome c heine lyase. Although import is shown as occurring at contact sites, it is possible that this protein can be imported through unattached outer membrane regions. (h) Cytochrome c. It is not known whether import of this protein takes place in unattached outer membrane regions, as shown, or at contact sites. At physiological salt concentrations, cytochrome c is bound to cytochrome oxidase. Inner membrane: (i) Subunit Va of cytochrome oxidase. (j) The ADP/ATP translocator. Outer membrane: (k) The MAS70 protein. (I) Mitochondrial porin.
Why is it important to distinguish between these two models~ If the conservative sorting hypothesis is correct, then the impressive advances in the field of bacterial protein export 15can be applied to studies of mitochondrial biogenesis. On the other hand, if certain sequences can act as stop-transfer sig-
454
nals in mitochondria, an understanding of this process would shed light on the nature of mitochondrial import sites 2 and possibly on the mechanisms of translocation arrest in other systems. Our investigations of c y t o c h r o m e s b 2 and c~ suggest that these proteins foJ!ow a stop-transfer pathway. The following
Targeting followed by assembly In the past, proteins have been assigned to the Various mitochondrial compartments ~in a somewhat arbitrary and inconsistent fashion. The reason is that the proteins of a given compartment may be imported by several different routes. For example, cytochrome cl is generally considered to be targeted to the intermembrane space TM and subunit IV of cytochrome oxidase (CoxlV) is regarded as a matrix-targeted precursor 17, yet both proteins end up tightly associated with the inner membrane. We propose that for studies of mitochondrial biogenesis, proteins should be classified according to the compartment to which they are initially targeted. The subsequent assembly of oligomeric complexes should be considered separately. A targeting mechanism may be used by _an entire class of precursors, whereas each protein has a unique assembly pathway. Intramitochondrial sorting is therefore a two-stage process, with different signals and catalysts required for each stage. In the example given above, cytochrome g is targeted to the intermembrane space and CoxIV is targeted to the matrix, and both of these proteins then assemble into complexes in the inner membrane. To apply this classification system, we suggest the following definitions: (1) A protein is targeted to the matrix if every residue of the mature polypeptide is exposed to the matrix at some time during import. (2) A protein is targeted to the intermembrane space if every residue of the mature polypeptide is exposed to the intermembrane space at some time during import. (3) A protein is targeted to the inner membrane if part of the mature polypeptide is translocated across the inner membrane at contact sites, and part of the mature polypeptide never crosses the inner membrane. (4) A protein is targeted to the outer membrane if part but not all of the polypeptide crosses the outer membrane.
TIBS 17 - NOVEMBER 1992
(a) Matrix 4-
4.
=
4.
4.
Intermembrane space
~ • I
Mature
4-
&
A
(b) 4. 4.
4.
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4-
4- 4-
4-
4- 4-
-
.
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-
=
MLKYKPLLKISKNCEAAILRASKTRLNTIRAY GSTVPKSKSFEQDSRKFITQSWTALRVGAILAATSSVAYLNWHNGQIDN
A
'
E...,
•
%
(c) -I-4-
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MFSNLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAM.... I
I
A
•
(d) 4-
4-
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4-4-
4-
MLGIRSSVKTCFKPMSLTSKRL ISQSLLAS K ....
A
& Figure 2
The presequences of cytochromes b 2 and cI contain bipartite targeting signals. (a) The general structure of a presequence with intermembrane space targeting information. The amino-terminal portion of the presequence resembles a matrix-targeting signal; the carboxy-terminal portion contains a hydrophobic stretch (represented by the black box) that is necessary for targeting to the intermembrane space. The presequence is cleaved twice, first by the soluble matrix processing protease (~) and then by a protease facing the intermembrane space (A). (b)Yeast cytochrome b2; (c) yeast cytechrome cl; both cytochromes conform to the scheme outlined in (a). The hydrophobic stretch in each presequence is underlined, and charged amino acids are indicated. The first cleavage site for cytochrome cI has not been identified, but it is probably within the region marked by the bracket. For the amino acids shown in bold, mutations have been isolated that weaken or inactivate the intermembrane space targeting signals (Ref. 29; E. Beasiey, unpublished). In the cytochrome b2 presequence, this targeting signal has been shown to include residues outside of the hydrophobic stretch. (d) The presequence of the iron-sulfur protein is also cleaved in two steps, first by the general matrix processing protease (A) and then by a different enzyme (A). Both processing events probably occur in the matrix25. It is thus unlikely that this presequence contains intermembrane space targeting information.
This simplified scheme assumes that ample, mouse dihydrofolate reductase during translocation through both mem- (DHFR) can be targeted to the matrix by branes at contact sites, the precursor the presequence of subunit 9 of the Fo polypeptide chain is shielded from the complex 19 (Fig. la), to the intermemintermembrane space. This assumption brane space by the presequence of may not always be correct 2,3J8. For pro- cytochrome cl TM (Fig. le), to the inner teins such as cytochromes b2 and c~, membrane by internal targeting sethe distinction between targeting to the quences in the ADP/ATP translocator 2° matrix and to the intermembrane space (Fig. lj), or to the outer membrane by is unambiguous only for a stop-transfer the amino-terminal portion of the mechanism; if these proteins followed MAS70 protein 21(Fig. lk). a re-export pathway, this set of definitions would have to be modified. Fig. The iron-sulfur protein of complex III is 1 illustrates the import pathways of sev- targeted to the matrix eral mitochondrial proteins, grouped acThe definitions given above are useful cording to the definitions given above. for distinguishing between proteins This classification system is con- that reach the same intramitochondrial sistent with the results of gene-fusion location by different mechanisms. For experiments, in which a precursor's tar- example, in complex III of the respiratory geting signal is tested for its ability to chain, cytochrome ci and the iron-sulfur direct a passenger protein to a specific protein both have their active sites at mitochondria] compartment. For ex- the external face of the inner mem-
brane 22. T h e iron-sulfur protein is first translocated completely into the matrix, and then inserts into complex IlI by a process that probably resembles the corresponding bacterial assembly reaction 9,12,23,24(Fig. ld). Thus for the ironsulfur protein the conservative sorting model is clearly appropriate. However, there is disagreement about whether this model can be extended to cytochrome 619'12-14 . At first glance, cytochrome cl and the iron-sulfur protein might be expected to use similar sorting mechanisms. The presequences of both proteins are cleaved in two steps upon import 16,24. However, only the cytochrome cl presequence contains a bipartite targeting signal (Fig. 2). The amino-terminal portion of the cytochrome cl presequence is cleaved off by the matrix protease, and the intermembrane space targeting
455
TIBS 17 - NOVEMBER 1 9 9 2
import of cytochrome b 2 into isolated mitochondria, a large percentage of the (a) S t o p - t r a n s f e r (b) C o n s e r v a t i v e s o r t i n g molecules were apparently present in the matrix TM. Second, when the matrixlocalized hsp60 chaperone was inactivated in a OM ......... : , OM temperature-sensitive yeast mutant, the intermediatesized form of cytochrome b 2 accumulated in the cells, suggesting that hsp60 function was required for reexporting cytochrome b 2 to the intermembrane space 23. We decided to re-examine these findings, since insights into the dynamic nature of the mitochondrial import machinery had revived our interest in IM IM the stop-transfer model 12. To analyse the topology of imported cytochrome b2, Figure 3 we selectively ruptured the Two models for the targeting of cytochromes b2 and c1 to the intermembrane space. The wavy line with mitochondrial outer mem'+' signs represents the amino-terminal part of the presequence, and the solid rectangle represents brane by osmotic shock, the intermembrane space targeting domain. OM, outer membrane; IM, inner membrane. (a) Stop transand then added protease fer. The intermembrane space targeting domain arrests translocation through the inner membrane. The to digest molecules that amino-terminal portion of the presequence is then cleaved off by the matrix protease. The mature domain of the precursor continues to cross the outer membrane to the intermembrane space, where the were outside the inner remainder of the presequence is removed by a second protease. The hsp60 chaperone should not be membrane. This method involved in this pathway; matrix ATP may or may not be required. (b) Conservative sorting. The precurdid not reveal any matrixsor is imported completely into the matrix, where it interacts with hsp60. The intermembrane space tarlocalized sorting intergeting domain is then recognized by a bacterial-type export machinery, which translocates the protein mediates 13. By contrast, in back across the inner membrane to the intermembrane space. Matrix ATP shoutd be required both for previous studies the mitothe initial import into the matrix and for release from hsp60. Two-step cleavage would occur as in the stop-transfer model. chondria were subfractionated with the detergent digitonin; after this treatdomain is removed by a second pro- act as a signal-anchor sequence, since the ment the imported intermediate form of tease at the outer face of the inner protein apparently does not integrate cytochrome b 2 behaved like a soluble membrane u,~6. By contrast, the pre- into the lipid bilayer of the inner mem- matrix protein ~4. Although we could sequence of the iron-sulfur protein brane 24. Moreover, there is no evidence reproduce this result, additional experseems to contain only matrix-targeting that this hydrophobic stretch acts as a iments indicated that the cytochrome b2 information (Fig. 2). The iron-sulfur sorting signal. For these reasons we intermediate was not actually located in protein belongs to a class of precursors suggest that it is misleading to describe the matrix~, but was bound to the inner whose presequences are cleaved twice the assembly of the iron-sulfur protein membrane and facing the intermemin the matrix by two different en- as 're-export'. Similar arguments apply brane space ~3. A likely explanation for zymes25; other examples include the to other polypeptides that assemble the aberrant behavior of this protein soluble matrix proteins ornithine trans- into the inner membrane after being im- during digitonin fractionation is that the cytochrome b 2 intermediate was not carbamylase and malate dehydrogenase. ported into the matrix 17,27. integrated into the lipid bilayer, and This two-step cleavage probably functherefore was solubilized at lower detions to ensure correct processing Cytochromes b2 and c~: stop.transfer or tergent concentrations than endogenous rather than to specify intramitochon- conservative sorting? drial sorting 26. Initial results from our laboratory were inner membrane proteins 13. It is still unresolved whether or not Thus unlike cytochrome cl, which is consistent with a stop-transfer mechan intermembrane space-targeted pre- anism for the targeting of cytochromes hsp60 is necessary for targeting to the cursor, the iron-sulfur protein is tar- b 2 and c1: during import of a precursor intermembrane space. Using the same geted to the matrix. This p~rotein then containing the cytochrome ci pre- temperature-sensitive mutant as previassembles into complex II[ by an sequence, no intermediate forms were ously, we were unable to confirm that unknown mechanism. Although the found inside the inner membrane ~2. inactivation of hsp60 prevents the matumature iron-sulfur protein contains a However, other groups subsequently ration of cytochrome b2. In our hands, hydrophobic stretch near its amino ter- reported two results that s u p p o r t e d t h e cytochromes b2 and ci were processed minus 22, this domain probably does not conservative sorting model. First, after normally when hsp60 was inactivated 13.
D'[I
"
/
l
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Moreover, cytochrome b 2 maturation was still observed after hsp60 had been depleted from yeast cells with the aid of a regulatable promoter (R. L. Hallberg, pers. commun.). Recent in vitro experiments provide a further test of hsp60 function. A fusion protein was constructed in which a large portion of the cytochrome b 2 precursor was joined to the amino terminus of DHFR28. During import, methotrexate was added to prevent passage of the DHFR domain across the outer membrane. Despite the methotrexate block, both cleavage reactions of the presequence took place, indicating that part of the precursor was exposed to the second processing protease in the intermembrane space. The conclusion from the conservative sorting model was that re-export could begin even though import across the outer membrane was incomplete; thus a species was formed that spanned three membranes. In support of this interpretation, the mature fusion protein was associated with hsp60 after the mitochondria were disrupted with digitonin 28. However, it was not rigorously excluded that binding to hsp60 took place after the digitonin treatment. According to the stop-transfer model, this fusion protein was arrested with its amino terminus in the matrix, its DHFR domain outside the outer membrane, and its cytochrome b 2 moiety facing the intermembrane space. Cleavage by the second processing protease would therefore generate a mature protein that spanned only the outer membrane. Such a species would be on a 'deadend' pathway, unable to finish translocation to the intermembrane space. Indeed, our preliminary results indicate that this protein cannot cross the outer membrane even when the methotrexate is removed 03. Glick, unpublished). Additional work is needed to distinguish between the different proposed topologies of this intermediate. The two models for intermembrane space targeting were also tested by the following approaches: (1) Since matrixlocalized ATP is essential for complete import across the inner membrane ~8 and for the release of proteins from hsp60 ~9, the conservative sorting model predicts that matrix ATP should be required for targeting to the intermembrane space (Fig. 3). In fact, fusion proteins containing the presequences of either cytochrome cl or cytochrome b 2 were imported to the intermembrane space and processed normally in ATP-
depleted mitochondria 13. With authentic cytochrome c~, depletion of matrix ATP had no effect either on import or on the complex maturation pathway of this protein 29. (2) According to the stoptransfer model, the intermembrane :space targeting domain arrests translocation at an early stage in the import reaction. In the conservative sorting model this targeting domain acts later in the pathway, after the precursor has been transported into the matrix. A kinetic experiment showed that translocation was arrested at an early step, as expected for a stop-transfer mechanism 03. Glick and K. Cunningham, unpublished). (3) It is possible that; depending upon the conditions, some proteins can use either a stop-transfer or a conservative sorting mechanism. To test this idea, we artificially introduced a polypeptide containing the cytochrome cl presequence into the matrix. This protein was not retranslocated to the intermembrane space, suggesting that mitochondria lack a signal sequencedependent export system for proteins imported from the cytoplasm 03. Glick and K. Cunningham, unpublished). While none of these experiments is conclusive by itself, we believe that the combined data strongly favor the stoptransfer hypothesis. However, a consensus on this issue is still lacking. Table I summarizes the experimental evidence for each of the two models, together with alternative interpretations. Since we have criticized the conservative sorting model, we should also discuss some weaknesses of the evidence for a stop-transfer mechanism. The apparent lack of an hsp60 requirement for targeting to the intermembrane space could simply mean that cytochromes b2 and c~ do not need to interact with this chaperone as they pass through the matrix. Moreover, our inability to detect matrix-localized intermediates may indicate that reexport is faster than import. If re-export can begin before import into the matrix is complete 28, then under normal conditions one might not observe intermediates that were entirely inside the inner membrane. It is even possible that import and re-export are obligatorily coupled, so that the protein we artificially introduced into the matrix would not have been a productive sorting intermediate. The simultaneous import-reexport hypothesis implies that an intermembrane space targeting domain can bind to hsp60 during import28; such an interaction could account for the trans-
location arrest observed in our kinetic study. These arguments do not explain why cytochrome ci still undergoes normal targeting and maturation in ATPdepleted mitochondria 29. However, it is conceivable that import into such mitochondria occurs by a non-physiological mechanism, since even matrix-targeted precursors accumulate between the two membranes when ATP levels are low TM.
Intermembrane space targeting signals One indirect argument against the stop-transfer model has been that several proteins with extended hydrophobic stretches are imported through both membranes into the matrix24,27. Our analysis of intermembrane space targeting signals provides a solution to this problem. A genetic study of the presequence of cytochrome b 2 (Fig. 2) confirmed that the hydrophobic stretch is important for targeting to the intermembrane space (E. Beasley, unpublished). Unexpectedly, the introduction of Pro residues into this stretch severely disrupted the targeting signal; for example, converting Leu62 to Pro inhibited targeting more effectively than placing Arg at the same position. Thus the function of this domain apparently depends both on its hydrophobicity and on its conformation. Efficient targeting of cytochrome b 2 to the intermembrane space also requires basic residues upstream of the hydrophobic stretch, and Glu at position +1 of the mature protein (Fig. 2). These data support the suggestion that intermembrane space targeting domains resemble bacterial signal sequences 9. Indeed, the second cleavage of the cytochrome b 2 presequence is catalysed by an enzyme that is related to bacterial leader peptidase 3°. However, such observations do not imply that sorting occurs in the same way in bacteria and in mitochondria. We suggest instead that prokaryotic-type targeting sequences have been adopted during evolution as signals for a uniquely eukaryotic stop-transfer pathway. Because these stop-transfer signals are relatively complex, a hydrophobic sequence can be present in a precursor without necessarily arresting translocation through the inner membrane.
Sorting to the outer and inner membranes Proteins are sorted to the outer membrane by many different mechanisms. The insertion of mitochondrial porin (Fig. 11) seems to require the common translocation machinery in the 457
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1992
Table I. Stop-transfer or conservative sorting? Evidence for stop-transfer
Criticisms
Evidence for conservative sorting
Criticisms
(1) No sorting intermediates were found in the matrix for either cytochrome b 2 or cytochrome
Re-export might be very rapid. Under some conditions, reexport may be simultaneous with import28.
(1) The intermediate form of cytochrome b 2 co-fractionated with soluble matrix proteins when mitochondria were treated with digitonin14.
(2) The matrix-localized hsp60 chaperone was not required for the maturation of cytochromes b2 and c1 in vivo 13.
These results do not match a previous observation, which suggested that hsp60 was necessary for cytochrome b2 maturation in vivo 23, Moreover, re-export might still be possible in the absence of hsp60.
The digitonin method seems to give misleading results, because the cytochrome b 2 intermediate is not integrated into the lipid bilayer of the inner membrane13,
(2) When hsp60 was inactivated in a temperature-sensitive yeast mutant, the intermediate form of cytochrome b2 was detected in the cells 23.
This in vivo effect was not observed in another study13. Moreover, inactivation of hsp60 did not prevent cytochrome b 2 maturation in vitro 23,
(3) When import into the matrix was prevented by depleting the mitochondria of ATP, several intermembrane space-targeted proteins were still imported and processed normally13'29,
This phenomenon could represent a non-physiological side pathway, since even matrixtargeted precursors reach the intermembrane Space in ATPdepleted mitochondria18.
(4) When an intermembrane space-targeted precursor was placed in the matrix by artificial means, it was not re-exported a.
This precursor reached the matrix . by an abnormal pathway, and thus may have been unable to interact with the re-export machinery.
(3) A protein containing the cytochrome b 2 presequence could be trapped with part of the protein outside the mitochondria and another part in the intermembrane space. After detergent solubilization, this protein was bound to hsp6028.
Association with hsp60 might have occurred after the detergent treatment. In addition, this trapped protein apparently is not a productive intermediate on the pathway of import to the intermembrane spaceb.
(4) Some proteins with extended hydrophobic stretches are imported into the matrix24'27.
(5) A functional intermembrane space-targeting domain in a precursor protein caused a translocation arrest at an early stage of importa.
If hsp60 bound to this targeting domain during import28, translocation might have been slowed down.
A hydrophobic stretch by itself does not necessarily function as an intermembrane space targeting signal. These signals consist of several domainsc.
(5) The iron-sulfur protein of complex III is imported completely into the matrix before assembling into the inner membrane12,24.
The iron-sulfur protein probably follows a different sorting pathway than cytochromes b 2 and c1. Unlike these two cytechromes, the iron-sulfur protein does not have an intermembrane space targeting domain in its presequence25.
Ci12,13,29.
aB. Glick and K. Cunningham, unpublished; bB. Glick, unpublished; °E. Beasley, unpublished.
outer membrane 31. The import receptor MOM19 associates with the outer membrane by interacting with other components of the translocation complex32; thus for this protein the targeting and assembly reactions may be identical. A second import receptor, called MAS70 in yeast, is targeted to the outer membrane by its amino-terminal domain, which includes an extended hydrophobic stretch that functions as a membrane anchor 33 (Fig. lk). In experiments with chimeric precursors containing the MAS70 hydrophobic stretch, it was observed that insertion into the outer membrane could occur in either orientation, depending upon the sequence located upstream of the membrane anchor 21. MOM72, the Neurospora homologue of MAS70, requires MOM19 for its insertion34. Perhaps the nkost unusual mechanism for outer membrane localization is that of monoamine o~xidase B: this protein contains a targeting signal near its carboxyl terminus 35, and membrane insertion in vitro requires ubiquitin 36.
458
As discussed earlier, a number of proteins are initially targeted to the matrix or intermembrane space before assembling into oligomeric complexes in the inner membrane. Proteins can also be targeted directly to this membrane. For example, the ADP/ATP translocator apparently moves from the inner membrane import channel into the lipid bilayer without passing through the matrix (Fig. l j); i.e. this protein follows a nonconservative sorting pathway9,27. Subunit Va of cytochrome oxidase (CoxVa; Fig. li) is synthesized with a transient presequence, but targeting to the inner membrane also requires a hydrophobic stretch near the carboxyl terminus of the mature protein 37. Although the import mechanism of CoxVa is not known in detail, this hydrophobic sequence might act as an internal stop-transfer signal. Both the ADP/ATP translocator and CoxVa follow at least part of the same pathway that is used by matrix-targeted precursors 31,37. An entirely different import mechanism may exist for subunit VI of
complex III, as this acidic protein lacks a typical matrix-targeting signal38. Conclusion
Eukaryotic ceils have developed a generalized system for transporting proteins from the cytoplasm into the mitochondrial matrix. Many proteins reach the other mitochondrial compartments by diverging from this matrix import pathway at different stages. Mitochondrial protein sorting thus seems to be an example of evolutionary opportunism; with individual proteins selectively using components of a common translocation machinery for their own unique sorting mechanisms. References 1 Glick, B. and Schatz, G. (1991) Annu. Rev. Genet. 25, 21-44 2 Glick, B., Wachter, C. and Schatz, G. (1991) Trends Cell Biol. 1, 99-103 3 Planner, N., Rassow, J., van der Klei, I. and Neupert, W. (1992) Cell 68, 999-1002 4 Stuart, R. A. and Neupert, W. (1990) Biochimie 72, 115-121 5 Dumont, M. E., Ernst, J. F. and Sherman, F. (1988) J. Biol. Chem. 263, 15928-15937
TIBS 17 - NOVEMBER
1992
6 Nicholson, D. W., Hergersberg, C. and Neupert, W. (1988) J. Biol. Chem. 263, 19034-19042 7 Dumont, M. E., Cardillo, T. S., Hayes, M. K. and Sherman, F. (1991) Mol. Cell. Biol. 11, 5487-5496 8 Hakvoort, T. B. M., Sprinkle, J. R. and Margoliash, E. (1990) Proc. Natl Acad. Sci. USA 87, 4996-5000 9 Hartl, F-U. and Neupert, W. (1990) Science 247, 930-938 10 Lill, R. et al. (1992) EMBO J. 11, 449-456 11 Hurt, E. C. and van Loon, A. P. G. M. (1986) Trends Biochem. Sci. 11, 204-206 12 van Loon, A. P. G. M. and Schatz, G. (1987) EMBO J. 6, 2441-2448 13 Glick, B. S. et al. (1992) Cell 69, 809-822 14 Hartl, F-U., Ostermann, J., Guiard, B. and Neupert, W. (1987) Cell 51, 1027-1037 15 Wickner, W., Driessen, A. J. M. and Hartl, F-U. (1991) Annu. Rev. Biochem. 60, 101-124 16 van Loon, A. P. G. M., Br~ndli, A. W. and
IMPRESSIVE PROGRESS towards an understanding of the forces that stabilize the folded structures of globular proteins has been described in recent reviews in T I B S ~ and elsewhere 2,3. For most of the past 30 years it has been commonly supposed that the hydrophobic interaction makes a prominent contribution to the stability of the native form, as proposed by Kauzmann4. However, it has recently repeatedly been claimed that the contrary is true ~ . That no consensus now exists is demonstrated by the fact that two highly respected reviewers have examined the same body of data and have drawn opposite conclusions 2,3. This review summarizes the way in Which these divergent opinions arose and scrutinizes some of the arguments for reversing Kauzmann's hypothesis, especially those based on observations of relative stabilities of proteins of differing hydrophobicity. After some deliberation, it then appears that all available data are quite consistent with the conclusion that structural reorganization of water adjacent to non-polar groups opposes unfolding, as originally proposed.
Protein unfolding and dissolution of liquid .... hydrocarbons in water Protein denaturation resembles the formation of aqueous solutions of simple hydrocarbons in that both involve N. Muller is at the Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
© 1992,ElsevierSciencePublishers, (UK)
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4056-4060 26 Isaya, G., Kalousek, F., Fento0, W. A. and Rosenberg, L. E. (1991) J. Cell Biol, 113,
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27 Mahlke, K. et al. (1990) Eur. J. Biochem. 192,
551-555 28 Koll, H. et al. (1992) Ceil 68, 1163-1175 29 Wachter, C., Schatz, G. and Glick, B.S. EMBO J.
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20-24 36 Zhuang, Z. and McCauley, R. (1989) J. Biol. Chem. 264, 14594-14597 37 Miller, B. R. and Cumsky, M. G. (1991) J. Cell Biol. 112, 833-841 38 van Loon, A. P. G. M. et al. (1984) EMBO J. 3,
1039-1043
Does hydrophobichydration destabilize protein native structures?
Water in the immediate vicinity of a non-polar solute has characteristically low entropy and high heat capacity at 25°C. Common opinion has been that the insolubility of such species is caused by thermodynamic changes associated with the formation of these layers of abnormal water, 'hydrophobic hydration'. Recently, however, it has been proposed instead that hydrophobic hydration favors solution of hydrocarbons, or hydrocarbon sidechains, in water and therefore promotes protein unfolding. It is argued here that available data do not convincingly support this hypothesis.
transfer of non-polar groups from a nonaqueous to an aqueous environment4,5,7,9. This is characteristically accompanied by a large increase in the heat capacitY, proportional to the surface area of the hydrocarbon solute 1° or, for a protein, to the non-polar area exposed to water upon unfolding u. I t is generally interpreted as a manifestation of special structural or thermodynamic properties of water that is in contact with a non-polar group. Formation of this special kind of water is called hydrophobic hydration, and the resulting thermodynamic changes, here labelled with the subscript hph, constitute the hydrophobic effect. Observation of hydrocarbon solutions provides an opportunity to study this effect with minimal
interference from other interactions. Transfer of an alkane molecule from the pure liquid (1) to a water solution (w) is opposed by a large increase in the unitary Gibbs energy4, AG0(I-+w)
=
AH0(1-+w)
= - RTlnX,
-
TAS0(1--+w) (1)
where X is the mole fraction in the saturated solution, AH° is the enthalpy change and AS° is the entropy change. Near room temperature, the dominant term is TAS° (1-+w), which is large and negative. It has been reported that for an assortment of hydrocarbons the transfer enthalpies and entropies at any (Kelvin) temperature are well represented by the following empirical equations9:
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