Putting energy
into mitochondrial
Ellen M. Beasley, Clemens The University
Wachter
protein
and Gottfried
import Schatz
of Basel, Basel, Switzerland
The import of proteins into mitochondria occurs in several steps. At least three of these steps require ATP and involve molecular chaperones. This energy requirement has served as a useful tool for elucidating the import pathways into the four mitochondrial compartments. Current
Opinion
in Cell Biology
this cleavage occurs is not yet known. The folded, mature proteins may then assemble into homo- or heterooligomeric complexes. At least in some cases, folding or oligomerization is mediated by the mitochondrial hsp60 chaperone.
Introduction The mitochondrial genome encodes only a few proteins. Mitochondrial biogenesis is therefore dependent on the import of nucleus-encoded, cytoplasmically synthesized proteins. This import process has been intensively studied, and many of the signals that target proteins to distinct locations within mitochondria have been identified [ 141. Recently there has also been progress in identifying components of the machinery that transports proteins to their correct intramitochondrial locations. These studies have shown that mitochondrial protein import is a multi-step process, and that some of the steps require energy. An electrochemical potential across the inner membrane is necessary for the initial insertion of presursor proteins into that membrane [l-5]. In addition, ATP is needed at several stages in the import pathway. In this review we summarize recent progress in understanding the energy requirements of mitochondrial protein import. We also show how this understanding has been exploited to gain further insight into the overall import process.
Major
import
Most import pathways for the other mitochondrial compartments share at least some steps with the general ‘matrix pathway’ outlined above. For example, insertion into the outer membrane is usually dependent on mitochondrial surface receptors and probably on cytosolit anti-folding proteins as well, but it does not require a potential across the inner membrane or the matrix-localized components of the import machinery [ 61. Similarly, some proteins transported to the intermembrane space or the inner membrane initially follow the matrix pathway, but are diverted from it before crossing the inner membrane [ 1,7**,8**]. The intermembrane space protein cytochrome c moves directly across the outer membrane, thereby bypassing all the energy requirements and catalysts mentioned above [ 91. It is not yet possible to make any general statements about import into the outer membrane, the intermembrane space, or the inner membrane; until more information is available, each protein should be considered as a separate case.
routes
A discussion of the role of ATP in mitochondrial protein import requires a brief review of the entire process [ 1,4]. Matrix-targeted precursors follow a common import route: after synthesis on cytoplasmic polysomes, precursors are prevented from premature tight folding in the cytoplasm by binding to one of several ‘anti-folding’ proteins, which include members of the heat-shock protein (hsp) 70 family. The precursors are recognized by receptors on the mitochondrial surface and are then translocated through both the outer and inner membranes at sites where the membranes are in close contact. On the matrix side of the inner membrane, the precursors bind to a mitochondrial hsp (mhsp) 70, from which they are released into the matrix in an incompletely folded state. With most precursors, the matrix-targeting signal is removed by a soluble protease; the stage at which
DHFRAihydrofolate
646
1992, 4:646-651
reductase; @
Current
Tracking
energy
requirements
A role of ATP in mitochondrial protein import was first suggested on the basis of experiments with intact yeast cells [lo], but firm proof for such a role has been difftcult to obtain, The experiments with intact cells did not allow a distinction between the requirements for ATP and a requirement for an electrochemical potential across the inner membrane. Subsequent experiments with isolated mitochondria were often perfomled under conditions that did not effectively deplete ATP either outside or inside the mitochondria. Moreover, the submitochondrial location of proteins imported into ATP-depleted mitochondria was not always determined. It is now clear that in some cases proteins thought to be imported into the
Abbreviations hsp-heat-shock Biology
down
Ltd
protein; ISSN
mhsp-mitochondrial
0955-0674
hsp.
Mitochondrial
matrix of ATP-depleted mitochondria were transported across the outer membrane, but became stuck across the inner membrane (see below). Because of these experimental pitfalls, the literature contains several erroneous reports of ATP-independent import routes. Now the fog has lifted somewhat and the energy-requiring steps of mitochondriaJ protein import are reasonably well defined. There is now general agreement that at least three steps of protein import into the mitochondrial matrix require ATP. One of these ATP-requiring steps takes place in the cytoplasm, and the other two occur in the mitochondrial matrix (Fig. 1). All of the known ATP-requiring steps involve interaction with heat-shock proteins that are thought to act as molecular chaperones.
The role of cytoplasmic
ATP
The requirement for ATP outside the mitochondria is at least partly accounted for by the interaction of precursors with cytosolic 70 k13 chaperone proteins [ 11,121. These proteins bind newly sythesized precursors, maintain them in a loosely-folded, import-competent conformation, and release them again by hydrolyzing ATP [ 131. The cytosol may also contain other ATP-dependent or independent ‘anti-folding’ proteins [ 141 that interact with mitochondrial precursors.
protein
import
Beasley, Wachter, Schatz
The requirement for ATP outside the mitochondria can be bypassed by presenting the precursor to mitochondda as incomplete chains [ 151 or after denaturation with urea ( [ 16,171; C Wachter, B Glick, unpublished data). In principle, a co-translational import mode also should not require extramitochondrial ATP (except for protein synthesis itself), because the precursor would not fold before its trans-membrane passage. The relative importance of co- and post-translational import into mitochondria has still not been determined. In a cell-free yeast system, import of several precursors is exceptionally efficient if the precursors are synthesized in the presence of mitochondria [ 18.1. On the other hand, a chimeric, at-t&al precursor composed of an amino-terminal, mitochondrial targeting signal fused to dihydrofolate reductase (DHFR) is imported primarily by a post-translational mechanism in rYzo import is blocked by methotrexate, which only binds to the fully folded DHFR domain [19-l. Import is most probably either co- or post-translational, depending on the type of precursor and the physiological conditions [20,21-1. Import of some precursors appears to be inherently independent of extramitochondrial ATP. These precursors include apocytochrome c [9], cytochrome c heme lyase [8-l, fusion proteins composed of DHFR attached to a matrix-targeting signal [22] and possibly cytochrome oxidase subunit Va [23-l. It is not clear how these precursors differ from those that require extramitochondrial
Cytosol
Fig. 1. A schematic Matrix
the energy-requiring import into the
diagram steps mitochondrial
illustrating of
protein matrix.
(a) The precursor reacts with cytosolic chaperone heat-shock protein (hsp) 70 and is released in an ATP-dependent reaction. sor across a membrane sor interacts
-
Imported A’l’
Membrane
precursor potential
protein
q
g mL?i
Translocation system
(b) insertion of the the inner membrane potential. (c) The with mitochondrial
precurrequires precurhsp
(mhsp) 70, which may provide for pulling precursors across ner membrane. (d) Interaction
the force the inof hsp60
with the precursor may mediate sor refolding or oligomerization
precurin some
cases.
647
648
Membranes
ATP; perhaps they can maintain a translocation-competent conformation without the aid of antifolding proteins, or they can be unfolded at the mitochondrial surface b). acidic phospholipids [24].
1331. Therefore, this chaperonin apparently does not catalyze translocation of precursors across the mitochondrial membranes, but mediates refolding and oligomerization of at least some imported precursors [ 27**,32,33].
The role of matrix
ATP requirements for import mitochondrial compartments
ATP
The transport of precursors across the inner membrane requires ATP in the matrix [22]. This effect is now thought to reflect the ATP requirement of mhsp70 in the matrix. During translocation, precursors interact with mhsp70, which probably provides the driving force for pulling the precursors across the inner membrane [2,25,26,27**]. One possibility is that the binding of several mhsp70 molecules is required to drive the import of a single polypeptide chain [ 21; ATP hydrolysis would then serve to release the bound mhsp70 chaperones for another round of import. Alternatively, import of a polypeptide chain might require multiple rounds of ATP hydrolysis by a single mhsp70 molecule. Although the evidence for the role of mhsp70 is veF suggestive, it is still not proven that the requirement for matrix ATP is due to mhsp70, or even that mhsp70 is necessary for import. Even though ATP does promote the binding of mhsp70 to precursors during transtocation [27**], it is possible that mhsp70 interacts with incom ing precursor polypeptides simply because they are not completely folded. There is a discrepancy between the phenotype of a temperature-sensitive mhsp70 mutant and the in llitro ATP requirements for two mitochondrial precursors: mitochondria containing the mutant mhsp70 protein are unable to import clqochrome c1 or the ADP-ATP translocator at the restrictive temperature [28], even though these proteins are imported normall!~ after the matrix has been depleted of ATP (see below). The observation that inacti\,ation of mhsp70 blocks im port could reflect either a direct or an indirect effect of the mutation. Further work is necessay to resolve this issue. In Esckrichia cofi, the hsp70 homolog, dnaK, functions together with the dnaJ protein, suggesting that yeast dnaJ homologs are involved in the cytoplasmic and mitochondrial hsp70-dependent steps. Two dnaJ homologs that may be involved in mitochondrial import have indeed been found in yeast [29**,30°,310], One of them (the product of the MAS5 gene) is located in the cytosol and functions in mitochondrial protein import in zliru [29**,30*]. The other one is apparently mitochondrial, although direct interaction with mhsp70 has not yet been demonstrated [ 3 1.1. The second ATP-requiring reaction in the matrix is catalyzed by hsp60, which is homologous to groEL of bacteria. Hsp60 interacts with incompletely folded precursors [32] after they have been released from mhsp70 [ 27**], When mutant yeast cells expressing a temperaturesensitive hsp60 protein are shifted to the non-permissive temperature, precursors are still imported into the mitochondrial matrix, but accumulate as insoluble aggregates
to the different
The evidence summarized above predicts that import into the outer membrane. the intermembrane space and the inner membrane requires onI!, extramitochondriial ATP whereas import into the matrix needs both extGiand intramitochondrial ATP. This prediction is indeed correct for precursors that reach their final destination by a direct route, but it titils for those that follow :I ‘detour’ pathways. The ADP-ATP translocator is inserted into the inner membr:une without ;I detour through the 11x1~ trix [31]: import of this protein requires only esternal ATP (C Wachter, B Glick, unpublished data). In contr;1st. cytochrome osidase subunit N [35] is first completeI) transported into the matrix before assembling with its partner subunits in the inner membrane. Although this protein is tightI>, associated with the inner membrane, it is initially targeted to the matrix, and therefore requires both intra- and extr~iiitochondrial ATP. The import pathways of c?%)chrome 2, and cytochrome 0, into the intermembrane sp:lce have been c‘ontro\‘ersial. According to the ‘stoptransfer’ hypothesis, these proteins :ire transported across the outer membrane. but are pre\rented from crossing the inner membrane by stop-transfer signals in their presrquences [36.3-l; according to the ‘consenXi\re sorting’ hypothesis. these proteins are first completely transported into the matrix, and are then re-exported across the inner membrane to the intermembrane space [3X]. Only. the conselv3tive sorting model predicts that import to the intermernbrane space requires ATP in the matrix. Recent studies show that import of cytochrome 2, to the intermembrane space of isolated mitochondria is independent of matrix ATP, suggesting that import occurs via a stop-transfer mechanism ( [?-*I; C Wa c.h ter. B Glick, unpublished data). Import of cytochrome h does require intramitochondrial ATP [ 221, but this requirement can be partially bypassed by denaturing the precursor with urea ( [?**I; C Wachter, B Glick, unpublished data). QTochrome c heme Iyase, which uses onl~~the outer membrane import machinery, also reaches the intermembrane space without passing through the matrix; import of this protein does not require intramitochondrial ATP [w].
Other
ATP-requiring
steps?
There is currently no evidence for additional ATP-requiring steps in mitochondrial protein import. It appears that the import machinery in the outer membrane does not use ATP. In Nezrrospora crassu, the MOM38 subunit of
Mitochondrial
the machinery has a weak consensus motif for ATP-binding [39]. In ISP42, the homologous yeast protein, this motif is only barely discernible [40], and its consensus amino acids can be replaced without inactivating the function of ISP42 in llivo (J Biosca, K Baker, unpublished data). In addition, proteins targeted to the matrix can cross the outer membrane in the absence of ATP ( [ 27.0,41**,42*]; see below). These results suggest that extramitochondrial ATP is needed only for the function of cytosolic anti-folding proteins. The three identified ATP-dependent steps are sufficient to explain the ATP requirements of all protein import pathways investigated so far. However, it is unclear whether there are additional ATP-requiring steps inside the mitochondria. Such steps could include chaperone-mediated assembly reactions in the intemiembrane space, or an energy-dependent transport reaction catalyzed by subunits of the import machinery in the inner membrane.
protein
Beaslev, Wachter, Schatz
import
[ 461. The import pathway of cytochrome c heme lyase to the intermembrane space is evidence that import through the outer membrane system can be uncoupled from im port through the inner membrane in vivo [Boa].
Conclusion Although there has been rapid progress in this field, many of the import pathways to the mitochondrial compartments are still incompletely characterized, and many components of the machinery involved have not yet been identified. The task ahead is daunting, but the recent advances reviewed here should be of great help in tackling it.
Acknowledgements The ‘ATP-depletion dissecting import
intermediate’: into the matrix
a tool for
The direrent energ)’ requirements of individual import steps halve been used to interrupt import at defned stages, to identie import catalysts and to deduce the order in which these catal!rsts operate. When ;I matrix-targeted precursor is added to ATP-depleted mitochondria that still maintain ;I membrane potential, the amino-terminal matrix-targeting signal penetrates across the outer membrane and inserts into the inner membrane. The rest of the Iprecursor chain initialI!. remains outside the outer membrane, but then moves slowl\~ across that membrane into the intermembrane S~XIW [ ;I**,tZ*] The resulting ‘ATP-depletion intermedi:ltc‘ is stuck in the inner mem brane: it can be chased into the nutris by restoring the ATP level in the matrix. This chase, \vhich is \-en’ rapid and independent of a membrane potential, seems to represent 3 partial reaction of the o\~ernll import process.
thank our cotitz~gues in the laboratoq’ for helpful suggestions and discu.sslcxx Our 0An work cited in this article was supported hy grams from the Sniss National Science Foundation (3.26189.89). the 1’5 Public Health Senice 12 ROI GM 378033 and the Human Frontier Science Program Organization. and by a long-term postdoctoral fellowship to EMB from the European Molecular Biology Organicttitxl.
\\‘e
References Palwr~ of \icn. have of of
and recommended
particular interest, published been highlighted as: special interest outstandmg mtewt
Guc:~ :l,r,~tr
B. SCII.UZ Kn, Ge,rcl
reading
nithin
the annual
G: Import of Proteins 1991, 25.2 1-t-1.
NEI’PERT W. Ii,wrI. Polypeptides Cross 1990. 63:++?+50.
FL‘. the
into
period
of re-
Mitochondria
Cwt. EA. PF~\N~XEH iv. How Mitochondrial Membranes?
BAK;EH K, S(:~LU~ G: Mitochondrial bility Mediate Protein Import ttrw 1991, 349~705-708.
into
Proteins Yeast
Essential for Mitochondria.
Do Cc/l Via1%
Additional stages of translocation into the matris were resol\.ed lq. manipulating both mitochondrial ATP levels and the folding state’ of a chimeric precursor protein [ -t3*]_ The ATP-depletion in:ermediate mnd its chase into the matrix have been used both to identik the cat$,sts that drive import across the inner membrane and to detine the earliest reactions of ;I precursor in the matris [27-l. The intermediate is also a promising substrate for crosslinking esperiments to idcntib. components of the import machineq in the inner membrane. Translocation intermediates that are arrested at various stages of import have alread!. been instiumentll in identif$ig mhsp70 [ 261 and components of the outer membrane clunnel (39,-tO.44,45*].
GIJW BS. BRANISI- A CtxstNcxw K. blt:u~lt 5. HXLWXC Rl, Sc~.lc!/) gene wzs cloned and sl1on.n optimal grcmzh.
of YQJl: a Yeast ./ Cc>// Rio/ 1991.
30. .
to he essential
for
BLM~HERG H. SIL\ZH PA: A Homologue of the Bacterial HeatShock Gene dnaJ that Alters Protein Sorting in Yeast. Nrclilt-e 199 I, .349:62’-630. This study identitird :I clnaJ-like !‘ti~st protein that appears to he at kXISt partly located in mitochondria and to affect intracellular protein sorting. 31. .
32.
OS~R.\WSN J. I IOHW’I~II AL. NEI’I’IXI’ W. I i.~ri. FL’: Protein Folding in Mitochondria Requires Complex Formation with hsp6O and ATP Hydrolysis. Na/lr,r 1989. 34 1: 125-l 30.
33.
CIWG MY, fH,WTI. FL’. ~LWIIS J. f’oI.I.0Cii K/I. fhlOl’~rl!K F. NEI‘IWT W, If,\ilI1ER(; E&l. lhu~i:RG RL. IfORwICIl AL Mitochondrial Heat-Shock Protein hsp60 is Essential for Assembly of Proteins Imported into Yeast Mitochondria. &arrrre 1989. 337:62ti25. f+ANNER N, Nelll’r:R’r W: Distinct Steps in the Import of ADP/ATP Carrier into Mitochondria. ./ Hrol C/XIII 19X’. 262:-‘528-‘536.
35.
HL’KT EC, PEsou).fit’R-r B. Sc~t:~iz G: The Cleavable Repiece of an imported Mitochondrial Protein is Sufficient to Direct Cytosolic Dihydrofolate Reductase into the Mitochondrial Matrix. FEHS Len 19%. 178:306310.
36.
\‘,a LOON APGM. SCI~A’L! G: Transport Mitochondrial Intermembrane Space: of the Cytochrome cl Resequence quence Specfic for the Mitochondrial Em0 J 19x7. 6:2+t-2+18.
37.
H~IRT EC, v&y IQON and lntramitochondrial 1986. 11:20+206
21. ..
22.
KRIEG
A Precursor Rotein Partly Trdnslocated chondria is Bound to a 70 kd Mitochondrial ElfBO J 1990, 9:1315-&322.
&ID GA SCHATZ G: Import of Proteins into Mitochondria: Extramitochondrial Pools and Post-Translational import of Mitochondrial Protein Precursors In Vivo. J Rio/ Chem 1982, 257:13062-13067.
SMIIH BJ. YAFFE MP: A Mutation in the Yeast Heat-Shock Factor Gene Causes Temperature-Sensitive Defects in Both Mitochondrial Protein Import and the Cell Cycle. ill01 Cell Biol 1991, 11~2647-2655. Protein import into mitochondria itz 1ti180 is inhibited by blocking transcription of heat-shock proteins. Evidence is presented that confirms that both co- and post.translational import occur it? cdtjo.
Conformational
25.
of rvrtrl
12.
16.
Outer Membrane Involves a Lipid-Mediated Change. .I Biol Chem 1988. 264:2951-2956.
of Proteins to the The ‘Sorting’ Domain is a Stop-Transfer SeInner Membrane.
APGM: How Proteins Compartments.
Find Mitochondria 7irprcls Hiochem
Sci
3%
HARTL FL’. N~rlrefcr W: Protein Sorting to Evolutionary Conservations of Folding Science 1990. 247:93@938.
Mitochondriaand Assembly.
39.
KIEBLEH M, PFALIVILR R. SOLLUER T, GIUFFITIIS G. HOKST~IANN H, PFANNER N, NEIII’ERI’ W: Identification of a Mitochondrial Receptor Complex Required for Recognition and Membrane Insertion of Precursor Proteins. Nulrrre 1990. 348:61&616.
Mitochondrial 40.
BAKER KP. SCHANIEL A, VES~WEBEK D, SCHA-IZ G: A Yeast Mitochondrial Outer Membrane Protein Essential for Protein Import and Viability. Nurure 1990, 348:605409.
41. . .
HWANG ST, WACHTER C, SCHA’U G: Protein Import into the Yeast Mitochondrial Matrix-A New Translocation Intermediate Between the Two Mitochondrial Membranes. J Biol Cktn 1991, 266:21083-21089. This study shows that depleting the matrix of ATP causes matrixtargeted precursors to accumulate as translocation intermediates between the two mitochondrial membranes, nith their amino.terminal domain stuck inro or across the inner membrane. 42. .
RASSOW J. PFANNER N: Mitochondrial Preproteins En Route from the Outer Membrane to the Inner Membrane are Exposed to the Intermembrane Space. Ff33S Leff 1991, 293:8WH An independent study identifying and chamcterizing the ‘ATP.depletlon inrermediate‘. 43. .
J~~CIIR T. G0IDENDERG DP, V~%TX’EHER D, SCIL\~L G: Sequential Translocation of an Artificial Precursor Protein Across the Two Mitochondrial Membranes. J Biol Chn 1992, in press.
protein
import
Beasley, Wachter,
Schatz
By manipulating a precursor‘s conformation and the ATP level in the matrix, a matrix.targeted precursor was first imported across the outer membrane and then across the inner one. 44.
VESIX’I?BER D, BRUNNER J, BAKER A, SCHATZ G: A 42 kd Outer Membrane Protein is a Component of the Mitochondrial Protein Import Site. Naiure 1989, 341:20>209
45. .
SOLINER T, &sow J. WIEDMANN M, SCHLosshtKvN J, P, NE~IPERT W. PFANNER N: Mapping of the Rotein port Machinery in the Mitochondrial Outer Membrane Crosslinking of Translocation Intermediates. Nalzrre 355:84-87. Components of the import machinery in the mitochondrial outer brane were identified by crosslinking them to a chimeric precursor tein that had been arrested during its tnnslocation across the membrane. 46.
KEIL Imby 1992, memproouter
GUCK B. WACHTER C, SCHATL G: Protein Import into Mitochondria: Two Systems Acting in Tandem? Trends Cell Rio1 1991. 1:9%103.
EM Bc~sley. C Wdchter center of the l’niversiv Switzerland.
and G Schatz, Department of Basel, Klingelbergstr.
of Biochemistry, 70, CH-4056
BioBasel,
651
into mitochondrial
Ellen M. Beasley, Clemens The University
Wachter
protein
and Gottfried
import Schatz
of Basel, Basel, Switzerland
The import of proteins into mitochondria occurs in several steps. At least three of these steps require ATP and involve molecular chaperones. This energy requirement has served as a useful tool for elucidating the import pathways into the four mitochondrial compartments. Current
Opinion
in Cell Biology
this cleavage occurs is not yet known. The folded, mature proteins may then assemble into homo- or heterooligomeric complexes. At least in some cases, folding or oligomerization is mediated by the mitochondrial hsp60 chaperone.
Introduction The mitochondrial genome encodes only a few proteins. Mitochondrial biogenesis is therefore dependent on the import of nucleus-encoded, cytoplasmically synthesized proteins. This import process has been intensively studied, and many of the signals that target proteins to distinct locations within mitochondria have been identified [ 141. Recently there has also been progress in identifying components of the machinery that transports proteins to their correct intramitochondrial locations. These studies have shown that mitochondrial protein import is a multi-step process, and that some of the steps require energy. An electrochemical potential across the inner membrane is necessary for the initial insertion of presursor proteins into that membrane [l-5]. In addition, ATP is needed at several stages in the import pathway. In this review we summarize recent progress in understanding the energy requirements of mitochondrial protein import. We also show how this understanding has been exploited to gain further insight into the overall import process.
Major
import
Most import pathways for the other mitochondrial compartments share at least some steps with the general ‘matrix pathway’ outlined above. For example, insertion into the outer membrane is usually dependent on mitochondrial surface receptors and probably on cytosolit anti-folding proteins as well, but it does not require a potential across the inner membrane or the matrix-localized components of the import machinery [ 61. Similarly, some proteins transported to the intermembrane space or the inner membrane initially follow the matrix pathway, but are diverted from it before crossing the inner membrane [ 1,7**,8**]. The intermembrane space protein cytochrome c moves directly across the outer membrane, thereby bypassing all the energy requirements and catalysts mentioned above [ 91. It is not yet possible to make any general statements about import into the outer membrane, the intermembrane space, or the inner membrane; until more information is available, each protein should be considered as a separate case.
routes
A discussion of the role of ATP in mitochondrial protein import requires a brief review of the entire process [ 1,4]. Matrix-targeted precursors follow a common import route: after synthesis on cytoplasmic polysomes, precursors are prevented from premature tight folding in the cytoplasm by binding to one of several ‘anti-folding’ proteins, which include members of the heat-shock protein (hsp) 70 family. The precursors are recognized by receptors on the mitochondrial surface and are then translocated through both the outer and inner membranes at sites where the membranes are in close contact. On the matrix side of the inner membrane, the precursors bind to a mitochondrial hsp (mhsp) 70, from which they are released into the matrix in an incompletely folded state. With most precursors, the matrix-targeting signal is removed by a soluble protease; the stage at which
DHFRAihydrofolate
646
1992, 4:646-651
reductase; @
Current
Tracking
energy
requirements
A role of ATP in mitochondrial protein import was first suggested on the basis of experiments with intact yeast cells [lo], but firm proof for such a role has been difftcult to obtain, The experiments with intact cells did not allow a distinction between the requirements for ATP and a requirement for an electrochemical potential across the inner membrane. Subsequent experiments with isolated mitochondria were often perfomled under conditions that did not effectively deplete ATP either outside or inside the mitochondria. Moreover, the submitochondrial location of proteins imported into ATP-depleted mitochondria was not always determined. It is now clear that in some cases proteins thought to be imported into the
Abbreviations hsp-heat-shock Biology
down
Ltd
protein; ISSN
mhsp-mitochondrial
0955-0674
hsp.
Mitochondrial
matrix of ATP-depleted mitochondria were transported across the outer membrane, but became stuck across the inner membrane (see below). Because of these experimental pitfalls, the literature contains several erroneous reports of ATP-independent import routes. Now the fog has lifted somewhat and the energy-requiring steps of mitochondriaJ protein import are reasonably well defined. There is now general agreement that at least three steps of protein import into the mitochondrial matrix require ATP. One of these ATP-requiring steps takes place in the cytoplasm, and the other two occur in the mitochondrial matrix (Fig. 1). All of the known ATP-requiring steps involve interaction with heat-shock proteins that are thought to act as molecular chaperones.
The role of cytoplasmic
ATP
The requirement for ATP outside the mitochondria is at least partly accounted for by the interaction of precursors with cytosolic 70 k13 chaperone proteins [ 11,121. These proteins bind newly sythesized precursors, maintain them in a loosely-folded, import-competent conformation, and release them again by hydrolyzing ATP [ 131. The cytosol may also contain other ATP-dependent or independent ‘anti-folding’ proteins [ 141 that interact with mitochondrial precursors.
protein
import
Beasley, Wachter, Schatz
The requirement for ATP outside the mitochondria can be bypassed by presenting the precursor to mitochondda as incomplete chains [ 151 or after denaturation with urea ( [ 16,171; C Wachter, B Glick, unpublished data). In principle, a co-translational import mode also should not require extramitochondrial ATP (except for protein synthesis itself), because the precursor would not fold before its trans-membrane passage. The relative importance of co- and post-translational import into mitochondria has still not been determined. In a cell-free yeast system, import of several precursors is exceptionally efficient if the precursors are synthesized in the presence of mitochondria [ 18.1. On the other hand, a chimeric, at-t&al precursor composed of an amino-terminal, mitochondrial targeting signal fused to dihydrofolate reductase (DHFR) is imported primarily by a post-translational mechanism in rYzo import is blocked by methotrexate, which only binds to the fully folded DHFR domain [19-l. Import is most probably either co- or post-translational, depending on the type of precursor and the physiological conditions [20,21-1. Import of some precursors appears to be inherently independent of extramitochondrial ATP. These precursors include apocytochrome c [9], cytochrome c heme lyase [8-l, fusion proteins composed of DHFR attached to a matrix-targeting signal [22] and possibly cytochrome oxidase subunit Va [23-l. It is not clear how these precursors differ from those that require extramitochondrial
Cytosol
Fig. 1. A schematic Matrix
the energy-requiring import into the
diagram steps mitochondrial
illustrating of
protein matrix.
(a) The precursor reacts with cytosolic chaperone heat-shock protein (hsp) 70 and is released in an ATP-dependent reaction. sor across a membrane sor interacts
-
Imported A’l’
Membrane
precursor potential
protein
q
g mL?i
Translocation system
(b) insertion of the the inner membrane potential. (c) The with mitochondrial
precurrequires precurhsp
(mhsp) 70, which may provide for pulling precursors across ner membrane. (d) Interaction
the force the inof hsp60
with the precursor may mediate sor refolding or oligomerization
precurin some
cases.
647
648
Membranes
ATP; perhaps they can maintain a translocation-competent conformation without the aid of antifolding proteins, or they can be unfolded at the mitochondrial surface b). acidic phospholipids [24].
1331. Therefore, this chaperonin apparently does not catalyze translocation of precursors across the mitochondrial membranes, but mediates refolding and oligomerization of at least some imported precursors [ 27**,32,33].
The role of matrix
ATP requirements for import mitochondrial compartments
ATP
The transport of precursors across the inner membrane requires ATP in the matrix [22]. This effect is now thought to reflect the ATP requirement of mhsp70 in the matrix. During translocation, precursors interact with mhsp70, which probably provides the driving force for pulling the precursors across the inner membrane [2,25,26,27**]. One possibility is that the binding of several mhsp70 molecules is required to drive the import of a single polypeptide chain [ 21; ATP hydrolysis would then serve to release the bound mhsp70 chaperones for another round of import. Alternatively, import of a polypeptide chain might require multiple rounds of ATP hydrolysis by a single mhsp70 molecule. Although the evidence for the role of mhsp70 is veF suggestive, it is still not proven that the requirement for matrix ATP is due to mhsp70, or even that mhsp70 is necessary for import. Even though ATP does promote the binding of mhsp70 to precursors during transtocation [27**], it is possible that mhsp70 interacts with incom ing precursor polypeptides simply because they are not completely folded. There is a discrepancy between the phenotype of a temperature-sensitive mhsp70 mutant and the in llitro ATP requirements for two mitochondrial precursors: mitochondria containing the mutant mhsp70 protein are unable to import clqochrome c1 or the ADP-ATP translocator at the restrictive temperature [28], even though these proteins are imported normall!~ after the matrix has been depleted of ATP (see below). The observation that inacti\,ation of mhsp70 blocks im port could reflect either a direct or an indirect effect of the mutation. Further work is necessay to resolve this issue. In Esckrichia cofi, the hsp70 homolog, dnaK, functions together with the dnaJ protein, suggesting that yeast dnaJ homologs are involved in the cytoplasmic and mitochondrial hsp70-dependent steps. Two dnaJ homologs that may be involved in mitochondrial import have indeed been found in yeast [29**,30°,310], One of them (the product of the MAS5 gene) is located in the cytosol and functions in mitochondrial protein import in zliru [29**,30*]. The other one is apparently mitochondrial, although direct interaction with mhsp70 has not yet been demonstrated [ 3 1.1. The second ATP-requiring reaction in the matrix is catalyzed by hsp60, which is homologous to groEL of bacteria. Hsp60 interacts with incompletely folded precursors [32] after they have been released from mhsp70 [ 27**], When mutant yeast cells expressing a temperaturesensitive hsp60 protein are shifted to the non-permissive temperature, precursors are still imported into the mitochondrial matrix, but accumulate as insoluble aggregates
to the different
The evidence summarized above predicts that import into the outer membrane. the intermembrane space and the inner membrane requires onI!, extramitochondriial ATP whereas import into the matrix needs both extGiand intramitochondrial ATP. This prediction is indeed correct for precursors that reach their final destination by a direct route, but it titils for those that follow :I ‘detour’ pathways. The ADP-ATP translocator is inserted into the inner membr:une without ;I detour through the 11x1~ trix [31]: import of this protein requires only esternal ATP (C Wachter, B Glick, unpublished data). In contr;1st. cytochrome osidase subunit N [35] is first completeI) transported into the matrix before assembling with its partner subunits in the inner membrane. Although this protein is tightI>, associated with the inner membrane, it is initially targeted to the matrix, and therefore requires both intra- and extr~iiitochondrial ATP. The import pathways of c?%)chrome 2, and cytochrome 0, into the intermembrane sp:lce have been c‘ontro\‘ersial. According to the ‘stoptransfer’ hypothesis, these proteins :ire transported across the outer membrane. but are pre\rented from crossing the inner membrane by stop-transfer signals in their presrquences [36.3-l; according to the ‘consenXi\re sorting’ hypothesis. these proteins are first completely transported into the matrix, and are then re-exported across the inner membrane to the intermembrane space [3X]. Only. the conselv3tive sorting model predicts that import to the intermernbrane space requires ATP in the matrix. Recent studies show that import of cytochrome 2, to the intermembrane space of isolated mitochondria is independent of matrix ATP, suggesting that import occurs via a stop-transfer mechanism ( [?-*I; C Wa c.h ter. B Glick, unpublished data). Import of cytochrome h does require intramitochondrial ATP [ 221, but this requirement can be partially bypassed by denaturing the precursor with urea ( [?**I; C Wachter, B Glick, unpublished data). QTochrome c heme Iyase, which uses onl~~the outer membrane import machinery, also reaches the intermembrane space without passing through the matrix; import of this protein does not require intramitochondrial ATP [w].
Other
ATP-requiring
steps?
There is currently no evidence for additional ATP-requiring steps in mitochondrial protein import. It appears that the import machinery in the outer membrane does not use ATP. In Nezrrospora crassu, the MOM38 subunit of
Mitochondrial
the machinery has a weak consensus motif for ATP-binding [39]. In ISP42, the homologous yeast protein, this motif is only barely discernible [40], and its consensus amino acids can be replaced without inactivating the function of ISP42 in llivo (J Biosca, K Baker, unpublished data). In addition, proteins targeted to the matrix can cross the outer membrane in the absence of ATP ( [ 27.0,41**,42*]; see below). These results suggest that extramitochondrial ATP is needed only for the function of cytosolic anti-folding proteins. The three identified ATP-dependent steps are sufficient to explain the ATP requirements of all protein import pathways investigated so far. However, it is unclear whether there are additional ATP-requiring steps inside the mitochondria. Such steps could include chaperone-mediated assembly reactions in the intemiembrane space, or an energy-dependent transport reaction catalyzed by subunits of the import machinery in the inner membrane.
protein
Beaslev, Wachter, Schatz
import
[ 461. The import pathway of cytochrome c heme lyase to the intermembrane space is evidence that import through the outer membrane system can be uncoupled from im port through the inner membrane in vivo [Boa].
Conclusion Although there has been rapid progress in this field, many of the import pathways to the mitochondrial compartments are still incompletely characterized, and many components of the machinery involved have not yet been identified. The task ahead is daunting, but the recent advances reviewed here should be of great help in tackling it.
Acknowledgements The ‘ATP-depletion dissecting import
intermediate’: into the matrix
a tool for
The direrent energ)’ requirements of individual import steps halve been used to interrupt import at defned stages, to identie import catalysts and to deduce the order in which these catal!rsts operate. When ;I matrix-targeted precursor is added to ATP-depleted mitochondria that still maintain ;I membrane potential, the amino-terminal matrix-targeting signal penetrates across the outer membrane and inserts into the inner membrane. The rest of the Iprecursor chain initialI!. remains outside the outer membrane, but then moves slowl\~ across that membrane into the intermembrane S~XIW [ ;I**,tZ*] The resulting ‘ATP-depletion intermedi:ltc‘ is stuck in the inner mem brane: it can be chased into the nutris by restoring the ATP level in the matrix. This chase, \vhich is \-en’ rapid and independent of a membrane potential, seems to represent 3 partial reaction of the o\~ernll import process.
thank our cotitz~gues in the laboratoq’ for helpful suggestions and discu.sslcxx Our 0An work cited in this article was supported hy grams from the Sniss National Science Foundation (3.26189.89). the 1’5 Public Health Senice 12 ROI GM 378033 and the Human Frontier Science Program Organization. and by a long-term postdoctoral fellowship to EMB from the European Molecular Biology Organicttitxl.
\\‘e
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