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ANRV313-BI76-30 ARI 8 May 2007 21:56
Translocation of Proteinsinto MitochondriaWalter Neupert1 and Johannes M. Herrmann2
1Institut fur Physiologische Chemie, Universitat Munchen, 81377 Munchen,Germany; email: [email protected] fur Zellbiologie, Universitat Kaiserslautern, 67663 Kaiserslautern, Germany;email: [email protected]
Annu. Rev. Biochem. 2007. 76:723–49
First published online as a Review in Advance onJanuary 30, 2007
The Annual Review of Biochemistry is online atbiochem.annualreviews.org
This article’s doi:10.1146/annurev.biochem.76.052705.163409
Copyright c© 2007 by Annual Reviews.All rights reserved
0066-4154/07/0707-0723$20.00
Key Words
chaperones, membranes, protein folding, protein import, TIM,TOM, translocases
AbstractAbout 10% to 15% of the nuclear genes of eukaryotic organisms en-code mitochondrial proteins. These proteins are synthesized in thecytosol and recognized by receptors on the surface of mitochondria.Translocases in the outer and inner membrane of mitochondria me-diate the import and intramitochondrial sorting of these proteins;ATP and the membrane potential are used as energy sources. Chap-erones and auxilliary factors assist in the folding and assembly of mi-tochondrial proteins into their native, three-dimensional structures.This review summarizes the present knowledge on the import andsorting of mitochondrial precursor proteins, with a special emphasison unresolved questions and topics of current research.
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Translocase: amembrane-embedded proteincomplex thatmediatestranslocation ofpolypeptides fromone side of themembrane to theother side
Contents
INTRODUCTION. . . . . . . . . . . . . . . . . 724MITOCHONDRIAL PRECURSOR
PROTEINS . . . . . . . . . . . . . . . . . . . . . 724Cytosolic Precursors . . . . . . . . . . . . . . 725Mitochondrial Targeting Signals . . 725
TRANSLOCATION OFPROTEINS INTO THEMATRIX . . . . . . . . . . . . . . . . . . . . . . . . 726The TOM Complex of the Outer
Membrane . . . . . . . . . . . . . . . . . . . . 726The TIM23 Translocase . . . . . . . . . . 728
IMPORT OF OUTERMEMBRANE PROTEINS. . . . . . . 732Topogenesis of Mitochondrial
Outer Membrane β-BarrelProteins . . . . . . . . . . . . . . . . . . . . . . 732
IMPORT OF INNER MEMBRANEPROTEINS . . . . . . . . . . . . . . . . . . . . . 734The TIM22 Pathway . . . . . . . . . . . . . 735The Stop-Transfer Pathway . . . . . . . 738The Conservative Sorting
Pathway . . . . . . . . . . . . . . . . . . . . . . 739PROTEIN IMPORT INTO THE
INTERMEMBRANE SPACE . . . . 739Proteins with Bipartite
Presequences . . . . . . . . . . . . . . . . . . 741Import by Folded Trap
Mechanisms. . . . . . . . . . . . . . . . . . . 742
INTRODUCTION
Almost the whole complement of proteinsthat constitute the mitochondria are encodedin the nucleus. These proteins are translatedon ribosomes in the cytosol as precursors,which differ from the functional forms in themitochondria in a number of ways. Most ofthem, if not all, are in an unfolded conforma-tion and associated with chaperones, whichmaintain them in a translocation-competentconformation. They are endowed with sig-nals for targeting to the surface of the mi-tochondria and for transport and sorting tothe various mitochondrial subcompartments.In order to become functional proteins, these
precursors have to be recognized by receptors,threaded through pores in the membranes ofthe mitochondria, proteolytically processed,folded and often inserted into membranes,and assembled with cofactors and other pro-teins to macromolecular complexes.
During the past 10 years, a large body ofinformation has been collected on the path-ways, and the translocases and protein compo-nents that mediate these processes. Both thenumber of pathways and the number of com-ponents discovered have grown beyond whatwas generally expected. We have now a muchmore detailed picture of how the plethora ofnuclear-encoded proteins are targeted to themitochondria and sorted to the various mito-chondrial subcompartments. The present re-view is based on an overview, which was pub-lished 10 years ago in the Annual Review ofBiochemistry (1). It is focused on the progressmade since then and can be read as a contin-uation of the previous review.
MITOCHONDRIAL PRECURSORPROTEINS
A large body of evidence suggests that mi-tochondrial proteins are synthesized on freeribosomes in the cytosol, released as com-pleted precursor polypeptide chains, and thenimported into mitochondria in a posttransla-tional fashion. Observations to support thisview have been obtained by experiments invivo and in vitro. This, however, does notmean that proteins cannot be imported co-translationally (1). Because most preproteinshave N-terminal targeting signals, transloca-tion could well start before the polypeptidechain is completed. In fact, in some cases, sucha mechanism has been proposed to explain theparticularities of the biogenesis of certain pro-teins such as fumarase (2, 3). It was proposedthat mechanisms exist that direct messengerRNAs to the surface of the mitochondriawhere they could be translated on ribosomesbound to the outer membrane (4, 5). Defi-nite proof for the existence of such targetingmechanisms, however, has not been provided.
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Cytosolic Precursors
Precursor proteins in the cytosol are presentas complexes with factors that are thoughtto stabilize them, as they are not in theirfinal conformation and therefore are proneto degradation and aggregation. Several suchfactors have been described; the only ones forwhich convincing evidence has been providedare cytosolic chaperones Hsp70 and Hsp90 (6,7). It is still unclear what the signals are thatlead to the binding of these chaperones, whenthey are bound and released, and whetherthere is a specific role for the targeting sig-nals present on the precursors.
Mitochondrial Targeting Signals
Cytosolic precursors of mitochondrial pro-teins contain information that is necessary andsufficient to direct them to the mitochondria.In most cases, this information is present asa cleavable sequence at the N terminus, alsocalled presequence or prepeptide. However,many precursors lack such sequences and in-stead contain internal targeting signals.
Matrix targeting signals. The N-terminaltargeting sequences are also called matrix-targeting sequences (MTSs) because they alsobring the N terminus across the inner mem-brane into the matrix. In the absence of fur-ther sorting information, they direct proteinsinto the matrix. They have been studied inconsiderable detail, and their main charac-teristics have been known for more than 10years. They consist of about 10–80 amino acidresidues that have the potential to form am-phipathic helices with one hydrophobic andone positively charged face. There is no con-sensus in the primary structure, which oftendiffers considerably even between closely re-lated orthologs. However, the general prop-erties of these amphipathic helices are widelyconserved among fungi and animals. In plants,the MTSs are still similar, although usuallylonger and richer in serine residues (8). Theconserved properties of MTS allow searches
PROGRAMS USED TO IDENTIFYMATRIX-TARGETING SEQUENCES
There are several prediction programs, based on different al-gorithms, that can identify mitochondrial proteins and distin-guish them from proteins of other cellular subcompartments.Some of these programs and their Internet addresses are listedhere.
Name Internet address ReferenceTargetP http://www.cbs.dtu.dk/services/TargetP/ (185)PSORT II http://psort.ims.u-tokyo.ac.jp/ (186)MITOPRED http://bioinformatics.albany.edu/
∼mitopred/(187)
MitoProt II http://ihg.gsf.de/ihg/mitoprot.html (188)Predotar http://urgi.infobiogen.fr/predotar/
predotar.html(189)
for it with prediction programs. Several ofthese programs are publicly available andlisted in the sidebar Programs Used to Iden-tify Matrix-Targeting Sequences.
N-terminal targeting signals are inmost cases cleaved from precursors by themitochondrial-processing peptidase (MPP)as soon as the cleavage sites reach the matrix(for reviews see References 9 and 10).
Preprotein: theprecursor form of amitochondrialpolypeptide, usuallyunfolded or looselyfolded, which oftencontains targetingsignals(presequences)
Presequence (orprepeptide): amitochondrialpreprotein’sN-terminalextension, oftenremoved by themitochondrialprocessing peptidase,followingtranslocation intothe matrix
Mitochondrial-processingpeptidase (MPP): amatrixmetalloprotease,consisting of twosubunits, αMPP andβMPP, that removespresequences frompreproteins
The N-terminal positioning of the MTS isimportant for its function. However, artificialtransplantation of an MTS to the C terminusof a protein still led to its translocation intomitochondria, but in the C to N direction (11).Remarkably, there exists a protein in yeast, theDNA helicase Hmi1, in which the MTS isnaturally present at the C terminus (12). Incontrast, the insertion of an MTS in the cen-tral regions of proteins did not lead to mi-tochondrial targeting. There is, however, theinteresting situation that some inner mem-brane proteins contain internal MTS-like se-quences, which are C terminal to hydrophobicstretches. These structures might form hair-pin loops that mimic the amphipathic struc-tures of a typical MTS. Examples are Bcs1,Tim14, and Mdj2.
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Translocase of theoutermitochondrialmembrane (TOM)complex: the outermembrane proteintranslocase thatfunctions as generalentry gate forpreproteins intomitochondria
TIM23 complex:the inner membranemultisubunit proteincomplex thatmediatestranslocation ofpreproteins acrossand into the innermembrane
Internal targeting signals. Many mito-chondrial precursors do not contain N-terminal presequences but instead haveinternal signals. These typically lack consis-tent patterns, and the nature of these sig-nals remains largely elusive. Internal signalsare present in proteins that are destined toall mitochondrial subcompartments. Precur-sors of outer membrane proteins all have in-ternal signals. Although the targeting signalsin β-barrel proteins remain unidentified, mi-tochondrial targeting of tail-anchored outermembrane proteins, such as bcl-2, Tom5,or Fis1, is dependent on the signal-anchordomain. A moderate hydrophobicity of thisregion and positive charges at the very Nterminus were found to be indicative forthese proteins and allow their distinction fromsignal-anchor proteins of the endoplasmicreticulum (13–16).
The internal signals that target proteins tothe intermembrane space (IMS) or the innermembrane are described in detail below.
TRANSLOCATION OFPROTEINS INTO THE MATRIX
The majority of mitochondrial proteins re-side in the matrix of the mitochondria. Theyare imported into this innermost subcom-partment by the cooperation of the maintwo mitochondrial preprotein translocases,the TOM complex in the outer membraneand the TIM23 complex in the inner mem-brane. These complexes interact with eachother to transfer the preproteins across bothmembranes in a concerted manner.
The TOM Complex of the OuterMembrane
The TOM complex is the translocase in theouter membrane that mediates the importof virtually all proteins of the mitochon-dria. It recognizes the precursor proteins inthe cytosol, facilitates release of cytosolic-binding factors, contributes to the unfold-ing of cytosolic protein domains, transfers the
polypeptides through pores across the outermembrane, and mediates insertion of someresident outer membrane proteins.
Architecture of the TOM complex. Thecomposition and structure of the TOM com-plex of various organisms have been investi-gated in some detail. Most of what is knownabout the TOM complex comes from studiesof the fungi Neurospora crassa and baker’s yeastwhere only very minor differences have beenobserved. Even in more distantly related or-ganisms, such as animals and plants, the struc-ture and function of the TOM complex ap-pears highly comparable to that in fungi (17–19).
Seven components were found to consti-tute the TOM complex (Figure 1). They canbe grouped according to their functions into
IMS
OM
Cytosol
Tom40Tom22Tom20Tom7Tom6Tom5
Surface receptorTranslocation poreSurface receptorSurface receptorTranslocation poreTranslocation poreTranslocation pore
Tom70
7 65
22 20
40
70
TOM complex
Figure 1Composition of the TOM complex. The TOM(translocase of the outer membrane) complexconsists of the receptor subunits Tom70, Tom22,and Tom20 and the membrane-embeddedsubunits Tom40, Tom7, Tom6, and Tom5, whichform the translocation pore. Abbreviations: IMS,intermembrane space; OM, outer membrane.
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receptor and pore components. Tom20 andTom70 are the major receptors that recog-nize preproteins. Both proteins are anchoredwith N-terminal transmembrane segments inthe outer membrane and expose hydrophilicdomains to the cytosol. Tom70 and Tom20differ in their substrate specificity, but bothreceptors overlap in their function and canpartly substitute for each other. The cytosolicdomain of Tom70 contains 11 tetratricopep-tide repeat motifs (20, 21), which show a sub-strate preference for hydrophobic precursorsthat contain internal targeting signals. Thesemotifs not only recognize the precursors, butalso interact specifically with cytosolic chap-erones, namely with Hsp70 and, in animals,with Hsp90. Binding of ATP then triggers therelease of the precursors from the chaperonesand their insertion into and passage throughthe TOM pore (7).
Tom20 is the main receptor for N-terminalpresequences. Structural analysis by NMR ofa part of the cytosolic domain of Tom20 incomplex with a prepeptide demonstrated abinding groove for the hydrophobic face ofthe MTS (22). In animals, Tom20 interactswith the cytosolic factor AIP. This proteincontains tetratricopeptide repeats and mayinteract with preprotein-binding chaperonesin a way similar to Tom70 (23).
Tom22 spans the outer membrane in anNout-Cin orientation. It exposes a highly neg-atively charged N-terminal domain to the cy-tosol and a short C-terminal domain to theIMS. Tom22 connects Tom20 to the centraltranslocation pore and may cooperate withTom20 in the binding and unfolding of pre-cursor proteins. In addition, Tom22 plays acritical role for the general integrity of theTOM complex (24, 25).
The general import pore of the TOMcomplex consists of the central compo-nent Tom40 and three small associated sub-units, Tom5, Tom6, and Tom7. Tom40 is amembrane-embedded protein that presum-ably forms a β-barrel structure. Even in theabsence of other TOM subunits, purifiedTom40 forms pores in artificial membranes
that show characteristics similar to that of theentire TOM complex (26–28). It is, however,not clear whether in the TOM complex thepores are formed by single or multiple Tom40molecules. The small TOM proteins consistof about 50 to 70 amino acid residues. They allform tail-anchored, probably α-helical struc-tures with only a few residues exposed to theIMS. The loss of individual small TOM pro-teins leads only to minor effects, but the si-multaneous deletion of all three proteins islethal (29–31). They appear to stabilize theTOM complex, yet their individual functionsare still unclear.
The TOM complex, when purified usingthe mild detergent digitonin, has a molecularmass of roughly 490–600 kDa (32–34). Single-particle imaging of a negatively stained TOMcomplex showed particles with two or threepore-like structures (Figure 2a). Purificationprocedures, using less gentle detergents likedodecylmaltoside, lead to the loss of the pe-ripheral receptors Tom70 and Tom22 andyield the TOM core complex of 350–450 kDa,which contains two pores of about 20 A in
Figure 2Electron micrograph of the TOM complex. Single-particle imagereconstitution after electron microscopic analysis and correlation averagingof negatively stained isolated TOM complexes represent: (a) the entireTOM complex (32), (b) the TOM core complex without Tom70 and Tom20(35), and (c) the homooligomeric Tom40 complex (34). Note that the holocomplex shows two or three pores, whereas the core complex contains two,and the Tom40 complex, only one pore. It was suggested that two of thepores in the holo complex represent aqueous translocation channels. Thethird cavity might represent peripheral receptor subunits associated to thecore complex. The bar corresponds to 5 nm. Reprinted with permission ofThe Journal of Cell Biology (34, 35) and of Elsevier for Cell (32).
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diameter (35) (Figure 2b). Purified Tom40on its own forms homooligomeric structures,which consistently show one cavity of dimen-sions similar to the TOM pores (Figure 2c).
Function of the TOM complex. The TOMcomplex contains several binding sites forprecursor proteins. These sites are presenton the cytosolic domains of Tom70, Tom20,and Tom22, which are often referred to ascis-binding sites. In addition, binding sitesare present on the IMS-exposed surface ofthe TOM complex. The nature of the trans-binding site is not entirely clear, but Tom22,Tom7, and Tom40 appear to contribute toprecursor binding in the IMS (36–38). Anincrease in the binding affinity presumablydrives the vectorial translocation of the pre-sequence across the outer membrane (39)and can facilitate even the unfolding of theprecursor protein (38, 40). Notably, the TOMcomplex can act in this way as a molecularchaperone. Precursors bound to the trans siteare protected against aggregation apparentlyby removal from the pool of unfolded speciesin the cytosol (38, 41, 42).
The TIM23 Translocase
The TIM23 complex is the major prepro-tein translocase in the inner membrane ofmitochondria. In cells that are highly ac-tive in oxidative metabolism, such as fungalcells or metazoan muscle cells, its client pro-teins may make up some 20% of total cellu-lar protein. The TIM23 complex translocatesall precursors of matrix proteins, most innermembrane proteins, and many proteins of theIMS. Translocation by the TIM23 complexis energetically driven by the electrical mem-brane potential across the inner membrane(�ψ) and the hydrolysis of ATP. The com-ponents of the TIM23 complex can be sub-divided into two groups, which likely oper-ate in a sequential and cooperative manner:(a) those that form the protein-conductingchannel (the membrane sector) and (b) those
IMS
IM
MatrixHsp70Mge1ATP
50
17
23
21
17
50
23
14 16 44
21
141644
ΔψΔψ
Tim23 Membrane sectorTim21 Membrane sectorTim17 Membrane sectorTim16/Pam16 Import motorTim14/Pam18 Import motormtHsp70 Import motorMge1 Import motor
Membrane sectorTim50Import motorTim44
-
+
TIM23 complex
Figure 3Composition of the TIM23 complex. Schematicrepresentation of the subunits of the TIM23translocase. Tim 21, Tim50, and Tim23 exposedomains to the intermembrane space (IMS), whichinteract with incoming polypeptides. Tim17 andTim23 are constituents of the translocationchannel. Tim44, Tim16, Tim14, Hsp70, andMge1 are associated at the matrix side. They makeup the import motor. Other abbreviations: �ψ,electrical membrane potential; IM, innermembrane.
that drive the translocation into the matrixspace (the import motor) (Figure 3).
The membrane sector of the TIM23complex. The membrane sector is composedof the three essential subunits, Tim50, Tim23,and Tim17, and a recently identified fourthprotein, Tim21, which is dispensable forTIM23 function (43). Tim23 and Tim17 formthe membrane-embedded core of the TIM23complex. Both proteins share a phylogeneti-cally related membrane domain consisting offour transmembrane segments. Additionally,Tim23 exposes an N-terminal hydrophilicregion to the IMS. In yeast, this region
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consists of two parts. The N-terminal partwas found to span the outer membrane, atleast under certain conditions, and might playa role in the positioning of the TIM23 translo-case in proximity to the outer membrane (44).The second part (residues 50–100) containsan essential coiled-coil domain, which is crit-ical for dimerization of Tim23 and for sub-strate binding (45–47). Recombinant Tim23showed conductances of various sizes between0.14 and 1.4 picosiemens (pS) after reconsti-tution into lipid membranes (48) and, hence,is able to form a channel; whether this chan-nel indeed represents the protein-conductingchannel of the TIM23 complex is not known.
The function of Tim17 is not clear. Incontrast to Tim23, Tim17 exposes only avery short N-terminal tail of about 11 to 14residues to the IMS. Nevertheless, this tailcontains conserved negative charges, whichare critical for preprotein import. Tim17 wasproposed to play a critical role in gating of theTIM23 pore (49).
Tim50 is anchored into the inner mem-brane by an N-terminal transmembrane do-main and exposes a large domain to the IMS(46, 47, 50). Tim50 interacts with incomingpolypeptide chains as they reach the trans siteof the TOM complex and presumably passesthem on to Tim23. Tim50 not only functionsas a passive import receptor but also seemsto play a critical role in the regulation of im-port channel’s permeability and, thus, mightcoordinate the translocation process of pre-proteins by the TIM23 complex (51).
Even less is known about the structure andfunction of Tim21, which was only recentlyidentified (43). Tim21 was observed to inter-act with the IMS domain of Tom22, suggest-ing a role in the interaction of the TIM23complex with the TOM complex (43, 52). An-other role of Tim21 was proposed, regulatingthe association of the import motor with themembrane sector of the TIM23 complex. Theexistence of two forms of the TIM23 com-plex was suggested, one with both the mem-brane sector and the import motor and an-other one without the import motor (43). The
first form, free of Tim21, may be specializedon translocation into the matrix, whereas thesecond one may occur on translocation of pre-cursors, which are subject to stop transfer. Itis important to further analyze the structureand composition of the TIM23 complex tohelp verify this interesting proposal.
The import motor. The membrane sectorof the TIM23 complex can transfer only theMTS of precursor proteins, a reaction drivenby the �ψ. Then the import motor has totake over. The following proteins play a rolein mediating the further vectorial movementof the unfolded polypeptide chain: Tim44,Tim14 (Pam18), Tim16 (Pam16), mtHsp70,and Mge1.
Tim44 is a hydrophilic matrix protein thatis completely (in fungi) or partially (in an-imals) attached to the inner membrane. Inyeast, a fraction of Tim44 can be coisolatedwith the membrane sector of the TIM23 com-plex (53, 54). The crystal structure of a C-terminal fragment of Tim44 showed a largehydrophobic pocket, which was proposed torepresent the membrane-binding site (55).Tim44 is in close contact with import inter-mediates and might function as a binding plat-form for motor subunits, bringing them inclose proximity to the incoming chains.
One interaction partner of Tim44 is theHsp70 chaperone of the matrix, mtHsp70. Aswith all Hsp70 chaperones, mtHsp70 consistsof an N-terminal ATPase domain and a C-terminal substrate or peptide-binding domain(56, 57). Hsp70s switch between ATP-bound,ADP-bound, and empty states. The ATP formhas an open binding pocket for substrates withhigh on and off rates. In the ADP form, thebinding pocket is closed, and the on and offrates are low. The nucleotide-free form is anintermediate in the cycle. The exchange ofADP by ATP occurs through the nucleotide-free state. This step requires the action ofthe nucleotide exchange protein Mge1, a ho-molog of the bacterial GrpE.
The ability of Tim44 to recruit mtHsp70is a key element in the import motor
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because it brings mtHsp70 to the very siteat which the precursor polypeptides appearin the matrix. Tim44 seems to recruit ATP-bound mtHsp70, which then can immedi-ately grasp the incoming, unfolded polypep-tide as its substrate-binding site is open. TheADP form appears to have a lower affinity forTim44. This suggests that upon hydrolysis ofATP mtHsp70 is released from the import site(58–60).
Substrate binding to Hsp70 chaperonesis typically regulated by DnaJ-like proteins.The import motor contains two subunits withDnaJ-like structures, Tim14/Pam18 (60–62)and Tim16/Pam16 (63–65). Tim14 containsan N-terminal membrane anchor that teth-ers the DnaJ-like matrix domain to the in-ner membrane. Cross-linking experiments in-dicate that Tim14 interacts with Tim44 andmtHsp70 in a nucleotide-dependent fashion:When ATP levels are low, Tim14 can becross-linked to Tim44; when they are high,Tim14 can be cross-linked to mtHsp70. Pre-sumably, Tim14 stimulates ATP hydrolysisin the mtHsp70 upon substrate binding andthereby triggers the release of the mtHsp70precursor complex from Tim44.
Tim14 forms a complex with the secondDnaJ homolog, Tim16. However, in contrastto Tim14, Tim16 is not a functional DnaJ-likeprotein because it lacks the HPD motif thatis critical for interaction with Hsp70. Consis-tently, Tim16 does not influence the ATPaseactivity of mtHsp70 in vitro and even counter-acts the ATPase-stimulating activity of Tim14(66, 67). The recently solved crystal struc-ture of the Tim14-Tim16 complex (68) re-vealed a tight interaction between both sub-units, in particular by a unique N-terminalarm on Tim14 that embraces Tim16. Dele-tion of this arm leads to a destabilization of thecomplex and is lethal in yeast. In the crystalstructure, the loop in Tim14, containing theHPD motif, is in direct contact with Tim16,fixed by several hydrogen bonds, and, thus,presumably unable to interact with Hsp70.This would confirm a function of Tim16 asa negative regulator of Tim14, which exhibits
its function by physically blocking the contactof Tim14 with mtHsp70.
Two additional components, Pam17 (69)and Mmp37/Tam41 (70, 71), were recentlyidentified, and their involvement in the as-sembly or function of the import motor wassuggested. The precise molecular role of theseproteins is unknown.
The mechanism of TIM23-mediatedtranslocation. A working model forTIM23-mediated preprotein import is pre-sented in Figure 4. Preproteins are directedfrom the TOM complex to the TIM23complex by binding the IMS domains ofTim50 and Tim23 (Figure 4, step 1). Thetranslocation of the MTS across the TIM23translocase requires �ψ either for providingenergy to transfer the positively chargedpresequence or for gating the TIM23 chan-nel, or for both processes (Figure 4, step2). In the matrix, Tim44 binds the incomingpreprotein and passes it on to mtHsp70 in theATP-bound state (Figure 4, step 3). Tim14stimulates ATP hydrolysis, leading to a tightbinding of mtHsp70 to the preprotein andto the dissociation of mtHsp70 from Tim44(Figure 4, step 4). At this stage, the prepro-tein can only slide into the matrix becausethe bound mtHsp70 prevents backsliding.Repetition of these trapping reactions leads tostepwise vectorial translocation of the wholepreprotein into the matrix (Figure 4, step 5).Facilitated by the nucleotide exchange factorMge1, mtHsp70 is released eventually fromthe preproteins, which then undergo foldingand assembly. The TIM23 complex forms anoligomeric structure so that more than onemtHsp70 molecule is positioned at the outletof the TIM23 channel. This is instrumentalfor rapid binding of mtHsp70 to incomingpreproteins (“hand-over-hand model”) (54).
A matter of debate has been the mechanismby which mtHsp70 generates the vectorialmovement of precursor proteins (72, 73). Twodifferent mechanisms have been proposed.The Brownian ratchet mechanism suggeststhat the precursors are subject to spontaneous
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Figure 4Stages of TIM23-mediated protein translocation. (a) Preproteins are directed by the intermembranespace (IMS)-exposed domains of Tim21, Tim50, and Tim23 (21, 50, and 23, respectively) to theprotein-conducting channel of the TIM23 translocase (step 1). (b) Upon gating of the TIM23 channel,the presequences reach the matrix in an electrical membrane potential (�ψ)-dependent step (step 2),(c) where they interact with the ATP-bound state of mtHsp70 (step 3). (d ) Upon ATP hydrolysis,mtHsp70 tightly binds the incoming chain and is released from the TIM23 complex (step 4).(e) Repeated mtHsp70-binding cycles drive complete translocation of the precursor protein. mtHsp70 isfinally released upon exchange of the bound ADP for ATP, a reaction stimulated by Mge1 (step 5). Inmost cases, the presequence is proteolytically removed by the mitochondrial-processing peptidase(MPP). Abbreviations: 14, Tim14; 16, Tim16; and 44, Tim44.
sliding reactions in the import machinery,consisting of TOM and TIM23 complexes(74). Binding of mtHsp70 would trap translo-cated segments because retrograde movementis blocked (75, 76). This ratchet-type mecha-nism then leads to a stepwise inward move-ment. The molecular motor in this mech-anism is energetically powered by the ATPhydrolysis that is needed in the reaction cy-cle for binding of the polypeptide chain tomtHsp70 and for release of mtHsp70 fromTim44.
The power stroke mechanism, by contrast,proposes that mtHsp70 acts as a lever arm,which mechanically pulls on the precursorchain by a conformational change that occursupon ATP hydrolysis (72, 77, 78).
In the past years, a large body of evidencehas provided support for the first mechanism,and there is little doubt that unfolded pre-
cursors can oscillate in the import machineryand be vectorially moved by trapping on thematrix side by mtHsp70 (59, 73, 79). More-over, the capacity of a domain to be importeddoes not depend on its global unfolding, butinstead, it is correlated with the ability to un-dergo limited unfolding of N-terminal seg-ments following the MTS (73, 80, 81). Forcesrequired to unfold domains, as measured byatomic force microscopy, were not related totheir capacity to be imported (82–84). Theseobservations are completely compatible witha ratcheting mechanism of the motor, and ev-idence for an active pulling of mtHsp70 isstill lacking. The possibility that mtHsp70 inthe import motor can exert a relatively mi-nor force, which is determined by the energyof ATP hydrolysis and the length of a possi-ble segment moved in the inward direction,cannot be excluded. Such a force most likely
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TOB/SAMcomplex: the outermembrane complexthat catalyzes themembrane insertionand assembly ofβ-barrel proteins
TOB: topogenesisof mitochondrialouter membraneβ-barrel proteins
would not be sufficient to mechanically pullin a folded polypeptide chain, such as a titindomain.
IMPORT OF OUTERMEMBRANE PROTEINS
The TOM complex does not only mediatetransfer of precursors across, but also mediatesthe insertion of proteins into the outer mem-brane. There are different classes of outermembrane proteins that, as far as we know,use varied pathways for the insertion process.
One class comprises the proteins of Tom70and Tom20, which are anchored in the outermembrane with a transmembrane helix closeto their N terminus. These precursors donot use the receptors, and competition ex-periments suggested they also do not usethe protein-conducting channel of the TOMcomplex (85). Still, these proteins need theTOM translocase, possibly because the com-plex can facilitate protein insertion at itsprotein-lipid interface (86).
The outer membrane in addition containsa number of proteins of different topologies,such as proteins with C-terminal anchors (i.e.,the small subunits of the TOM complex) ora pair of internal transmembrane domains(i.e., Fzo1). Whether the TOM complex isinvolved in the binding or insertion of theseproteins is not known.
A particularly interesting class is theβ-barrel membrane proteins. Their in-sertion into the membrane requires thefunction of both the TOM complex andthe translocase of outer membrane β-barrel proteins (TOB)/sorting and assem-bly machinery (SAM) complex, a translocasespecialized in this process.
Topogenesis of Mitochondrial OuterMembrane β-Barrel Proteins
The outer membranes of mitochondria andchloroplasts are the only membranes in eu-karyotic cells that contain β-barrel proteins.This is likely to reflect the evolutionary ori-
gin of these organelles from bacterial ances-tors. So far identified β-barrel proteins inmitochondria are porin (or VDAC), Tom40,Tob55, Mdm10 and Mmm2. β-barrel pro-teins contain multiple membrane-spanningβ-strands each formed by 9–11 amino acidresidues (87). Precursors of β-barrel proteinsare imported via the TOM complex but re-quire a second outer membrane complex. Thisrecently identified translocase is named TOB(topogenesis of mitochondrial outer mem-brane β-barrel) (88) or SAM (sorting and as-sembly machinery) (89) complex.
Composition of the TOB complex. Themain component of the TOB complex isTob55, which was also named Sam50 orTom50 (Figure 5). It was identified bythree independent approaches: first, by ap-plying proteomic and biochemical analysis ofN. crassa mitochondrial outer membranes(88); second, by copurification with Mas37(90), previously assigned to the TOM com-plex of yeast and found to be involved in thebiogenesis of Tom40 (89); and third, by se-quence similarity to the bacterial Omp85 pro-tein (91). Tob55 consists of two domains: TheN-terminal hydrophilic region is exposed tothe IMS and forms a characteristic structure,called the POTRA (polypeptide translocationassociated) domain (92). The C-terminal do-main presumably forms a β-barrel domainwith 14 - 16 transmembrane β sheets. Ho-mologs of Tob55 are ubiquitously found ineukaryotes where they are present in the outermembranes of mitochondria and chloroplasts.They are also found in the outer membrane ofgram-negative bacteria, and they are referredto as Omp85 or YaeT (93, 94). Both the N-terminal POTRA domain and the C-terminalβ-barrel region are conserved among bac-terial and eukaryotic homologs, which havebeen implicated consistently in the biogene-sis of β-barrel proteins.
In addition to Tob55, the TOB com-plex contains two hydrophilic subunits, whichare located at its cytosolic surface. Theseproteins are Mas37 (Sam37/Tom37) and
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Figure 5The topogenesis of mitochondrial outer membrane β-barrel proteins (TOB) complex mediatesmembrane insertion of β-barrel proteins (88, 89). (a) Precursors of β-barrel proteins are translocatedacross the outer membrane (OM) by the TOM complex (step 1), (b) bound by small Tim proteins in theintermembrane space (IMS) (step 2) and (c) inserted and assembled by the TOB complex of the outermembrane (step 3). Synonyms of TOB components are shown in the inset. Abbreviation: SAM, sortingand assembly machinery.
Tob38 (Sam35/Tom38) (89, 95–97). Bothproteins have clear homologs only in fungi,but it was suggested that the mammalianprotein metaxin-1 represents a distant rela-tive of Mas37 (98). However, evidence fora Mas37-like function of metaxin-1 is stilllacking.
Tob55 and Tob38 are essential for viabil-ity of yeast cells. Depletion of these proteinsselectively blocks the insertion and assemblyof newly imported β-barrel proteins (88, 90,91). In contrast, Mas37 is largely dispensable,but mutants show defects in the biogenesisof β-barrel proteins at elevated temperatures.The specific functions of the three TOB sub-units are not clear, but the high conservationof Tob55 suggests that it is the central func-tional unit of the complex and that the othersubunits have rather an accessory function.
Topogenesis of mitochondrial β-barrelproteins. Precursors of β-barrel proteins in-teract with the receptors of the TOM com-plex, mainly Tom20, and presumably movethrough the TOM pore to insert into theouter membrane from the IMS site (Figure 5,step 1). This is indicated by the observationthat clogging the TOM channel by arrestingmatrix-targeted precursors blocked the im-port of β-barrel proteins (88, 89). In the IMS,complexes of small Tim proteins (see be-low) may guide these precursors from theTOM to the TOB complex (Figure 5, step2). Import across the TOM channel and in-sertion by the TOB machinery are function-ally coupled because when precursors wereimported into mitochondria lacking Tob55,they did not reach the IMS but accumulatedin the TOM complex. The POTRA domain
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of Tob55 may be involved in the recognitionof β-barrel precursors and might pass themon to the membrane-embedded C-terminalregion of Tob55, which then facilitates mem-brane insertion and assembly of the β-barrelproteins (Figure 5, step 3). The directionof insertion from the IMS into the outermembrane presumably reflects the evolution-ary origin of mitochondria from bacteria andthe assembly process of β-barrel proteins isstill exhibited by a phylogenetically old ma-chine in principally the same manner as inprokaryotes.
IMPORT OF INNER MEMBRANEPROTEINS
The inner membrane of mitochondria har-bors a large variety of different membraneproteins. Although some of these proteinsare synthesized by mitochondrial ribosomes,most inner membrane proteins are importedfrom the cytosol. Three different importroutes to the inner membrane were identified.They are depicted in Figure 6. First, innermembrane proteins with internal “hydropho-bic loop” signals use a specialized translo-cation complex in the inner membrane, the
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Figure 6Sorting pathways of inner membrane proteins. Proteins are sorted to the inner membrane by severalpathways, which differ with regard to the components involved and the direction of insertion. (a) Solutecarriers and hydrophobic TIM (translocase of the inner mitochondrial membrane) subunits are insertedinto the inner membrane by a dedicated inner membrane complex, the TIM22 translocase. Small Timproteins in the intermembrane space (IMS) presumably function as chaperones, which guide thehydrophobic precursors across the IMS. (b) Inner membrane proteins, which contain only onetransmembrane span, are arrested at the level of the TIM23 complex. They are integrated into the innermembrane by lateral exit. (c) A class of inner membrane proteins are sorted via soluble translocationintermediates in the matrix. Membrane insertion occurs here from the matrix side and, at least in certaincases, is facilitated by the Oxa1 (oxidase assembly) complex of the inner membrane.
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TIM22 complex. Second, preproteins can bearrested at the level of the TIM23 complexand laterally integrated into the inner mem-brane; this transport route is referred to as thestop-transfer pathway (99, 100). Third, someinner membrane proteins are initially translo-cated into the matrix, and from there they areinserted in an export-like step into the innermembrane. Because the direction of the inser-tion reflects that of the prokaryotic ancestorsof mitochondria, this pathway was called theconservative sorting pathway (101, 102).
The TIM22 Pathway
The Tim22 or carrier pathway employs threemitochondrial protein complexes that medi-ate in a successive order of steps the importand membrane insertion of the solute car-rier family members and of hydrophobic TIMsubunits (Figure 7). These complexes are theTOM complex in the outer membrane (seeabove), complexes of small Tim proteins inthe IMS, and the TIM22 translocase in theinner membrane.
Small Tim proteins. In the IMS, the smallTim proteins comprise polypeptides of 8 to12 kDa, which are characterized by a central“twin Cx3C motif,” i.e., two short motifs eachcomprising a pair of cysteine residues thatare separated by three residues. The regionsflanking this characteristic signature form α-helices, leading to a helix-loop-helix organi-zation of these proteins. Small Tim proteinsform hexameric structures of about 70 kDa insize. Structural analysis of the Tim9·10 com-plex revealed a ring-like organization in whichalternating Tim9 and Tim10 subunits are ar-ranged like the blades of a propeller (103–105). The core of this structure is formedby the central loop domains of the subunitsand is surrounded by 12 arms formed by theα-helical termini of the six subunits. Thus,the Tim9·10 complex resembles a flattenedjellyfish-like structure with 12 rather flex-ible tentacles. It was speculated that thesearms can accommodate the hydrophobic re-gions of carrier proteins in the IMS, thereby
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Figure 7Protein insertion by the TIM22 pathway.(a) Substrates of the TIM22 pathway are bound inthe cytosol by chaperones (stage1) and(b) recognized by receptors of the mitochondrialsurface (stage 2). (c) The preproteins then traversethe outer membrane through theprotein-conducting channel of the TOM complex(stage 3). (d ) In the intermembrane space (IMS),small Tim proteins guide the precursors to theinner membrane (stage 4). (e) There they areinserted by the TIM22 translocase in an electricalmembrane potential (�ψ)-dependent manner(stage 5). The membrane-embedded Tim22subunit of the TIM22 complex presumably servesas the central component of this translocase and isassisted by the accessory subunits Tim54 andTim18. Abbreviation: IM, inner membrane.
TIM22 complex:an inner membraneprotein complex thatmediates theinsertion of carrierproteins and subunitsof the TIMcomplexes
TIM: translocase ofthe innermitochondrialmembrane
shielding them from unproductive interac-tions (104). The structure to the Tim9·10complex bears some resemblance to the chap-erones prefoldin and Skp (106–108). Skp isa component of the bacterial periplasm thatguides β-barrel proteins from the inner to theouter membrane. This function is analogousto the function of small Tim proteins, which
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also interact with β-barrel proteins (109–111).Whether this similarity in function and struc-ture originates from a common phylogeneticancestry of both components is unknown. Al-though the idea of the small Tim proteins be-ing jellyfish-like chaperones that shield hy-drophobic regions of their substrates is in-triguing and attractive, it should be stressedthat at present this is still a hypothetical.The molecular mechanisms by which smallTim proteins fulfill their function in proteintranslocation are still largely unclear.
The twin Cx3C motifs are of special rel-evance for the formation of the structure ofsmall Tim complexes, and the mutation ofindividual cysteine residues is sufficient toblock complex assembly (112, 113). The cys-teine residues can form pairs of intramolec-ular disulfide bridges in which the two cen-tral and the two distal cysteines are bonded(114–116). By contrast, in their reduced state,small Tim proteins can bind zinc ions, pre-sumably by forming a zinc finger-like struc-ture (112, 117, 118). Indeed, trapping experi-ments under anaerobic conditions revealed areduced state of the cysteine residues (118).The observations on the oxidized or reducedstates of the cysteine residues are apparentlycontradicting, but it is conceivable that thestate of the cysteine residues is not perma-nently fixed. For example, during the biogene-sis of small Tim proteins, the cysteine residuesmay initially bind zinc ions before hexamer-ization of the monomers induces formationof disulfide bridges (119). Indeed, in the ab-sence of zinc ions, Tim10 rapidly oxidizes un-der the cytosolic redox conditions and therebyloses its import competence. Binding of zincprevents this oxidation and thereby facilitatesimport of Tim10 (119). A change in the re-dox state of small Tim proteins might, how-ever, not be limited to their biogenesis, butrather the complexes might permanently cy-cle between reduced and oxidized states (120,121). The IMS protein Hot13 was implicatedas a critical factor in the redox regulation ofsmall Tim proteins (121). However, knowl-edge about the specific function of Hot13 for
the redox dynamics of small Tim proteins isstill scarce, especially because in vitro systemsto analyze their activity with natural substrateshave not yet been developed.
The TIM22 complex. The TIM22 translo-case is a 300-kDa complex that consists of thethree membrane proteins Tim22, Tim54, andTim18 with which the three small Tim pro-teins, Tim9, Tim10, and Tim12, are associ-ated. Tim22 is related in amino acid sequenceto Tim17 and Tim23 (122). It represents theessential core of the complex that can medi-ate the insertion of carrier proteins even inthe absence of Tim54 and Tim18, although atstrongly reduced levels (123). Tim54 appearsto be an important, but not essential, acces-sory component of the TIM22 complex (123,124). Tim18 is a distant homologue of subunit3 of the succinate dehydrogenase, Sdh3, and,to date, has been found only in fungal mito-chondria (125, 126). The precise functions ofTim54 and Tim18 are not known.
A complex consisting of the small Timsubunits Tim9, Tim10, and Tim12 is per-manently tethered to the IMS side of theTIM22 complex. Tim54 might contribute tothe binding of this Tim9·10·12 complex be-cause its association with Tim22 was destabi-lized in tim54 deletion mutants (123). Tim12is an essential protein, and the Tim9·10·12complex may play a vital role for substraterecognition by the TIM22 complex.
Precursors using the TIM22 pathway.The TIM22 pathway is a sorting route se-lectively used by members of the solute car-rier family and by the membrane-embeddedTIM subunits Tim17, Tim22, and Tim23.All known substrates of the TIM22 com-plex represent membrane proteins with even-numbered transmembrane segments that ex-pose their N and C termini into the IMS.
Members of the carrier protein fam-ily consist of three pairs of transmembranesegments, forming sequence-related internalrepeats or modules (127–129). The targetinginformation is deciphered at three levels of the
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import reaction: at the surface of the mito-chondria to mediate binding to the Tom70receptor, in the IMS to bind to the Tim9·10complex, and at the level of the inner mem-brane for insertion by the TIM22 translo-case. Peptide-binding scans suggested that theregions around the hydrophobic transmem-brane domains are preferentially recognizedby Tom70 (130, 131) and the Tim9·10 com-plex (115, 132). Each of the modules appearsto contribute to these two initial recognitionevents. Import experiments with truncatedcarrier proteins suggested that each individ-ual module contains targeting informationfor translocation into the IMS (127, 133). Incontrast, only certain modules appear tomediate productive insertion into the in-ner membrane. In the ATP/ADP carrier andthe dicarboxylate carrier only the most C-terminal module contains the informationnecessary and sufficient for membrane in-tegration (127, 133). The molecular natureof the insertion signal in the third moduleremains still elusive.
Protein import by the TIM22 pathway.The TIM22 pathway can be divided intoseveral steps (134) (Figure 7). Followingtheir synthesis, carrier proteins are bound bythe cytosolic chaperones, Hsp70 and Hsp90(stage 1). These complexes bind to the re-ceptors of the TOM complex (stage 2) (7,135). In mammals, Hsp90 and Hsp70 dockonto the tetratricopeptide repeat domain ofTom70. Hsp90 thereby appears to play an ac-tive role in the translocation of carrier pro-teins across the TOM channel because thebinding of Hsp90 to Tom70 is vital for theimport of carrier proteins into mitochondria.In yeast, Hsp70 rather than Hsp90 is used inimport, and Hsp70 docking is required for theformation of a productive complex of prepro-teins with Tom70 (7).
Carrier proteins are then delivered to theTOM channel (stage 3) where they can ac-quire a topology in which the N and C ter-mini remain exposed to the cytosol (128, 136).These translocation intermediates interact in
the IMS with Tim9·10 complexes, which pre-sumably shield the hydrophobic domains ofcarrier proteins and accompany them acrossthe IMS from the TOM to the TIM22 com-plex (115, 117, 137–139). Finally, carrier pro-teins are taken over by the TIM22 com-plex and inserted into the lipid bilayer of theinner membrane in a membrane potential-dependent reaction (stage 4). Following theirrelease from the TIM22 complex, the carrierproteins assemble into their dimeric nativestate (stage 5). This dimerization occurs veryrapidly and is stimulated by the hydrophilichead groups of phosphatidylcholine, whichinduce a structural rearrangement in thecarriers (140, 141).
The import process of the TIM subunitsTim17, Tim22, and Tim23 is less well charac-terized than that of carrier proteins but seemsto adhere to the same principles (142–146).For its biogenesis, Tim23 makes use of an al-ternative small Tim complex in the IMS, con-sisting of Tim8 and Tim13 subunits. In con-trast to Tim9 and Tim10, Tim8 and Tim13are not essential components in yeast, pre-sumably because the Tim9·10 complex canfunctionally replace the Tim8·13 complex tosome degree.
In humans, loss-of-function mutationsof the Tim8 homologue DDP1 (deafnessdystonia polypeptide 1) cause the Mohr-Tranebjaerg syndrome (OMIM 304700), a re-cessive X-linked neurodegenerative disorder,characterized by hearing loss, cortical blind-ness, dystonia, mental retardation, and para-noia (147, 148). How mutations in DDP1interfere with mitochondrial function is notentirely clear. DDP1 appears to play a rolein the import of human Tim23 (113, 146,149) and also in the import of human car-rier proteins citrin and aralar1 (150). Bothproteins contain extended hydrophilic re-gions at their N termini. Tim23 also con-tains a long N-terminal hydrophilic region,and it was proposed that the Tim8·13 com-plex (or DDP1·Tim13 complex in humans)interacts specifically with substrates that con-tain hydrophilic extensions. Consistent with
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this idea, the Tim8·13 complex appears tospecifically interact with the N-terminal hy-drophilic region of the Tim23 precursor,whereas the Tim9·10 complex interacts withthe hydrophobic loops of the membrane-embedded region of Tim23 (145).
The Stop-Transfer Pathway
Many proteins of the inner membrane aresynthesized with typical MTSs. Most ofthem contain only one transmembrane do-main and adopt an Nin-Cout topology inthe inner membrane. Subunit 5a of the cy-tochrome oxidase of budding yeast (Cox5a)was one of the first representatives of thisclass whose biogenesis was studied in de-tail (99, 151, 152). Other proteins that ap-pear to be imported by the same principleare Cbp4, Cox11, Dld1, Hem14, MWFE,Mia40, Oms1, Pet117, Qcr7, Sco1, Sco2,She9/Mdm33, Tim50, Yme1, and Yme2.
Import experiments with purified mito-chondria suggested that Cox5a is inserted intothe inner membrane by a stop-transfer mech-anism, i.e., the transmembrane domain func-tions as a critical sorting signal that causesthe arrest of the precursor during the importreaction at the level of the inner membraneand inserts it laterally into the lipid bilayer.The import reaction is mediated by the TOMand TIM23 translocases. Upon deletion of itstransmembrane domain, Cox5a is mistargetedto the matrix (99, 153). The import of Cox5a,similar to that of matrix proteins, is driven bythe membrane potential and the mtHsp70-mediated hydrolysis of ATP. However, in con-trast to matrix proteins, Cox5a can be im-ported into mitochondria of mutants in whichthe function of mtHsp70 is impaired (152).When the transmembrane segment of Cox5a,which normally is placed in the C-terminalthird of the protein, was moved to the N ter-minus right after the presequence, ATP andmtHsp70 became completely dispensable forimport (151). In this case, the insertion intothe lipid bilayer apparently prevented back-sliding of the polypeptide.
Topogenic signals in stop-transfer pro-teins. The transmembrane segments of pre-cursors that use the stop-transfer pathwayhave to be recognized by the TIM23 translo-case. The mere presence of a hydropho-bic segment, however, is not sufficient fortranslocation arrest. Precursors of conserva-tively sorted proteins, despite the presence oftransmembrane segments in their sequences,do not become arrested but rather pass thoughthe TIM23 complex into the matrix. Stud-ies with mutant preproteins revealed at leastthree characteristic properties of arrested pro-teins that play a role as topogenic signals thatare deciphered by the TIM23 translocase:
1. Arrested transmembrane segments aretypically followed at their C terminiby clusters of charged amino acidresidues (154). The relevance of thesecharges was studied in some detailwith the D-lactate dehydrogenase ofyeast, Dld1. A sequence of six residueswith two positively and three nega-tively charged residues (Arg-Glu-Thr-Lys-Glu-Asp) is critical for transloca-tion arrest and deletion of this stretch,which caused mistargeting of Dld1 tothe matrix. Similar clusters of chargedresidues are often found in proteins thatare inserted on a stop-transfer route(153). Conceivably, the TIM23 com-plex interacts with these charges or withthe increased diameter of the polypep-tide chain because increased hydrationof the charged regions slows down theimport process and facilitates lateral in-sertion of the transmembrane domain.
2. The transmembrane spans of arrestedpreproteins, on average, are more hy-drophobic than those of conservativelysorted proteins. Especially tyrosine andphenylalanine residues are relatively en-riched in arrested sequences, whereasserine, threonine, asparagine, and glu-tamine residues are more abundantin transferred transmembrane domains(153).
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3. Proline residues within the trans-membrane domains are importantdeterminants for intramitochondrialsorting (153). Proline residues are typi-cally absent in arrested transmembranedomains but often are present in trans-ferred domains. The insertion of a pro-line residue into the transmembrane do-main of Cox5a caused the mistargetingof the protein to the matrix. By contrast,the removal of proline residues from atransmembrane domain of the conser-vatively sorted protein Oxa1 led to ar-rest of the Oxa1 precursor upon importand mediated its insertion into the innermembrane.
Stop-transferred proteins with Nout-Cin
topology. Although most precursors under-going stop-transfer are arrested with the Nterminus facing the matrix (Nin-Cout), someproteins reach the reverse orientation (Nout-Cin). These proteins typically contain aninternal transmembrane span C-terminallyfollowed by an amphipathic helix with ahydrophobic and a positively charged face. Itwas proposed that, during the import process,this sequence forms a hairpin-like structure,which resembles the structural features of apresequence, to promote protein import ona TIM23-dependent import route (155). Ex-amples are the AAA protein, Bcs1, and theJ-proteins, Tim14 and Mdj2.
The Conservative Sorting Pathway
Among the proteins that are synthesized withpresequences are several inner membraneproteins with more than one transmembranedomain. Examples of this group are subunit9 of the FoF1-ATPase of N. crassa, Oxa1,Cox18/Oxa2, Mrs2, and Yta10 (156–159).These proteins are initially translocated to thematrix where they are bound by mtHsp70.From the matrix, they integrate into the innermembrane in an export-like reaction, which isstill ill defined. At least in vivo, import into thematrix and insertion into the inner membrane
Oxa1 complex: aprotein complex thatfunctions in theinsertion ofmembrane proteinsfrom the matrix intothe inner membrane
can be separated into two subsequent reac-tions, suggesting that they form functionallyindependent processes (156, 157, 160). Theprotein segments that are transferred acrossthe inner membrane in most cases show a biasfor negatively charged residues. Membraneinsertion strongly depends on the membranepotential, presumably because the transferof the negatively charged regions to thepositively charged IMS drives the insertionreaction (156, 157, 160). The Oxa1 complexof the inner membrane facilitates the inser-tion of at least some of these inner membraneproteins (161, 162). Oxa1 belongs to a largefamily of proteins with members in mitochon-dria, chloroplasts, and bacteria (for a review,see References 163 and 164). In all these sys-tems, the members of the Oxa1 family mediatethe membrane insertion of proteins.
The term “conservative sorting” was ini-tially coined for the sorting pathway of theRieske iron sulfur protein (101). This proteinconsists of a large C-terminal IMS domainthat contains the iron sulfur cluster. In vitroimport experiments indicated that the entirepreprotein is initially imported into the ma-trix (165). There, the iron sulfur cluster of theprotein appears to be incorporated before thecatalytic domain, presumably in a folded con-formation, is exported into the IMS, and is as-sembled into the bc1 complex. Novel evidencefor a conservative sorting of the Rieske pro-tein recently came from genetic experimentsin which transfer of the gene to the mitochon-drial genome was shown to result in a func-tional protein that is properly placed into thebc1 complex (166). The mechanism by whichthe Rieske protein is exported across the innermembrane is still completely unknown.
PROTEIN IMPORT INTO THEINTERMEMBRANE SPACE
Proteins of the IMS have essential functionsin a variety of different processes, such asin metabolic and bioenergetic reactions, inthe transfer of metabolites and proteins be-tween the outer and the inner membrane,
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Cytosol
IMS
Matrix
IM
OM
Me
SSSS
ATP
Hsp70
TOMcomplex
TOMcomplex
TOMcomplex
TIM23complex
Bipartite presequences Folding traps Affinity sitesa b c
Figure 8Protein translocation into the intermembrane space (IMS). Three classes of IMS proteins can bedistinguished on the basis of their import mechanisms. (a) Proteins with bipartite presequences embarkon a stop-transfer route to the inner membrane; the mature parts of these proteins are then released intothe IMS by proteolytic cleavage. (b) Translocation of a class of small proteins is achieved by their folding,mediated by IMS-located factors after transfer across the TOM pore; trapping in the IMS makestranslocation vectorial. Formation of disulfide bridges (SS) or binding of metal cofactors (Me) occurs inmany cases. (c) A class of proteins requires binding to affinity sites in the IMS for net translocation.Abbreviations: IM, inner membrane; OM, outer membrane.
and in the control of regulated cell death. Al-though the function of many of these pro-teins was studied in detail, knowledge of theirbiogenesis is still scarce. On the basis oftheir import characteristics, most IMS pro-teins can be sorted into one of three categories(Figure 8):
1. Some IMS proteins contain a canon-ical N-terminal MTS, followed by ahydrophobic sorting sequence. Theirimport is mediated by the TOM andTIM23 complexes and depends on themembrane potential across the innermembrane and, in most cases, on ATP.Such bipartite presequences are re-moved by proteolytic processing releas-ing the mature proteins into the IMS(Figure 8a).
2. Many, if not most, IMS proteins areof low molecular weight, and their nettranslocation across the TOM complexrequires their folding in the IMS. Thefolding is triggered by the acquisitionof cofactors or by intramolecular disul-fide bridges. According to the foldingtrap hypothesis, the folded state pre-vents back-translocation out of the mi-tochondria and thereby confers unidi-rectional net import of these proteins(Figure 8b).
3. Some IMS proteins are permanently as-sociated with components in the IMS,and the affinity to these factors appearsto drive the import reaction (Figure 8c).Examples are the import of cytochromec heme lyase or creatine kinase.
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In the following section, specific represen-tatives of the first two of these classes will bedescribed in more detail.
Proteins with Bipartite Presequences
Proteins with bipartite presequences are re-leased into the IMS after proteolytic cleav-age. Although the targeting of these proteinsappears to adhere to a consistent principle,a surprising variety of processing peptidasesis employed to mediate proteolytic release ofthese IMS proteins. Some proteins and theirprocessing sites are sketched in Figure 9.Among these proteins are many of the apop-totic components that are contained in theIMS [similar to the apoptosis-inducing fac-tor (AIF)], i.e., HtrA2, Endonuclease G, andSmac/Diablo (Figure 9a). The import pro-cess of Smac/Diablo was characterized re-cently (167). Smac/Diablo contains a classi-cal mitochondrial presequence, followed by ahydrophobic sequence. Following import bythe TOM and TIM23 translocases, the hy-drophobic sequence is arrested at the levelof the inner membrane and laterally inte-grated into the lipid bilayer. This sorting
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 9Preproteins with bipartite presequences.Schematic representation of different precursorforms. Shown are the membrane-inserted sortingintermediates. Cleavage sites are indicated byarrowheads. (a) For most proteins, the bipartitepresequence is removed by cleavage at theintermembrane space (IMS) close to the innermembrane. (b) In the case of cytochrome cperoxidase (CCPO), the m-AAA protease removesan N-terminal part of the protein and contributesto the further translocation of the intermediate,allowing its cleavage by Pcp1. (c) Two alternativeforms of Mgm1 are produced by alternativeprocessing of different insertion intermediates.Abbreviations: AIF, human apoptosis-inducingfactor; CPO, coproporphyrinogen oxidase; Cyt.b2, cytochrome b2; HtrA2, human proapoptoticserine protease Omi/HtrA2; IM, inner membrane;m-AAA, m-AAA protease; Mcr1, cytochrome b5reductase; Mgm1, mitochondrial dynamin-likeprotein; NDPK, nucleotide diphosphokinase.
intermediate, which resembles an inner mem-brane protein in Nin-Cout topology, is recog-nized by the inner membrane peptidase (IMP)complex, which separates the transmembraneanchor from the C-terminal mature part of
AIFCPOHtrA2NDPK
Cyt. b2 Mcr1
Mgm1(precursor)
Mgm1(IM form)
Mgm1(IMS form)
CCPO
MPP
Pcp1
m-AAA
Pcp1
? Imp1
MPP
Imp1
IMS
Matrix
IM
c
b
a
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Smac/Diablo. The IMP complex contains thetwo proteolytically active subunits, Imp1 andImp2, which are related to bacterial leaderpeptidases (168).
Some proteins employ more than one pro-cessing peptidase. In the case of cytochromeb2, the mitochondrial targeting is cleaved byMPP in the matrix before the mature region ofthe protein is released into the IMS by cleav-age of the Imp1 protease. The cleavage byMPP therefore is not a prerequisite for importof cytochrome b2 because mutants in whichthe MPP processing site was deleted were stillproperly sorted to the IMS. In contrast, bothprocessing peptidases appear to play a cru-cial role for the import of yeast cytochromec peroxidase (CCPO) (169–171). CCPO con-tains an N-terminal mitochondrial targetingsequence, followed by two hydrophobic se-quences (Figure 9b). After its integrationinto the inner membrane, the N-terminalhydrophobic sequence is cleaved by the m-AAA protease, an ATP-hydrolyzing metallo-protease (172). The CCPO intermediate isthen further imported and matured by a sec-ond cleavage event, catalyzed by the rhom-boid protease Pcp1. Pcp1, in a way similar tothe m-AAA protease, cleaves its substrates athydrophobic transmembrane regions. In thecase of CCPO, this cleavage occurs at the sec-ond hydrophobic region and thereby releasesthe mature CCPO protein into the IMS. Thepresence of the m-AAA protease is essentialfor the import of CCPO, potentially becausethis oligomeric ATP-consuming enzyme iscritical for pulling the tightly folded IMS do-main of CCPO across the outer membrane.
Even more complex is the topogene-sis of Mgm1, a dynamin-related compo-nent of the mitochondrial fission machin-ery (173). Mgm1 is present in mitochondriain two differentially processed isoforms (seeFigure 9c): The short variant of Mgm1 isan IMS protein comprising residues 161–902of the Mgm1 precursor; the long variant isanchored to the inner membrane and com-prises residues 81–902 of the precursor. Bothvariants are produced in comparable amounts
by a process called “alternative topogene-sis.” The N terminus of Mgm1 contains twohydrophobic sorting sequences. For genera-tion of the long isoform, the N-terminal hy-drophobic sequence is inserted into the in-ner membrane from which the mitochondrialtargeting sequence is removed by MPP. Inabout half of the precursor proteins, the N-terminal hydrophobic sequence fails to inte-grate into the membrane and is transferredfarther into the matrix until the second hy-drophobic region arrests translocation. Thisregion is then cleaved by Pcp1, and the shortisoform is released to the IMS. The ratiobetween both variants depends on the ener-getic state of the mitochondria. Increased lev-els of ATP thereby promote the transfer ofthe N-terminal sorting sequence in the matrixand, thus, the generation of the short isoform.By contrast, depletion of ATP leads to in-creased levels of the long isoform. It was pro-posed that in vivo this is used to measure theATP levels in mitochondria. Mgm1 regulatesmitochondrial fusion and content mixing, aswell as segregation of mitochondrial genomes.Therefore, this mechanism might favor thepropagation of highly energetic mitochondriarelative to mitochondria harboring mutationsin their mitochondrial DNA.
Import by Folded Trap Mechanisms
Many proteins of the IMS are small, and theirfolding is stabilized by cofactors or disulfidebridges. The best-characterized representa-tive of this class is cytochrome c. The apo-form of cytochrome c is imported by the TOMcomplex into the IMS, where it is transientlybound by cytochrome c heme lyase (174–176).This enzyme inserts the heme cofactor intocytochrome c, which then is released in itssoluble holoform into the IMS. Thus, thebidirectional movement of the apoform ofcytochrome c across the TOM complex is con-verted to a vectorial import process. The en-zymatic activity of cytochrome c heme lyaselocks cytochrome c in its folded state andthereby traps it stably in the IMS.
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A similar trapping mechanism is used byother IMS proteins, such as the small Timproteins and proteins containing “twin Cx9Cmotifs.” This process relies on the formationof intramolecular disulfide bridges. Follow-ing their translocation across the outer mem-brane, the precursor forms are bound by theinner membrane protein Mia40 (177–179). Inits active state, Mia40 contains intramoleculardisulfide bridges that, upon interaction withprecursor proteins, are converted to inter-molecular disulfide bonds, covalently linkingthe precursors to Mia40. The imported pro-teins are finally released from Mia40, presum-ably in an oxidized state. The activity of Mia40relies on the function of Erv1, a conservedessential sulfhydryl oxidase in the IMS (180–182). This FAD-binding protein directly in-teracts with Mia40 and maintains it in an ox-
idized, active state. At present, it is not com-pletely clear how Erv1 itself is oxidized, butthe human homologue of Erv1 was efficientlyoxidized in vitro by oxidized cytochrome c(183). Cytochrome c might connect the elec-tron flow from oxygen and cytochrome c oxi-dase to Erv1 (181).
Cu,Zn-superoxide dismutase (Sod1) is an-other IMS protein that requires the bindingof metal cofactors for uptake, and the forma-tion of an intramolecular disulfide bridge inthe IMS is critical for its uptake by mitochon-dria (184). Both steps are catalyzed by theSod1-specific factor in the IMS, the copperchaperone of Sod1(CCS), which forms a tran-sient disulfide bridge with Sod1. This reac-tion is highly reminiscent of that occuring be-tween Mia40 and newly imported small Timproteins.
SUMMARY POINTS
1. Mitochondrial preproteins are synthesized in the cytosol with specific targeting sig-nals. In precursors of matrix-targeted proteins, these signals are mostly N-terminalpresequences, which are proteolytically removed from the proteins by a protease inthe matrix.
2. Receptors on the surface of mitochondria recognize preproteins.
3. The TOM complex mediates the transfer across the outer membrane.
4. β-barrel proteins are integrated into the outer membrane by the TOB complex.
5. The TIM23 complex contains the protein-conducting channel and the import motor,which together mediate the translocation of preproteins into or across the innermembrane.
6. The TIM22 translocase inserts carrier proteins and subunits of the TIM complexesinto the inner membrane.
7. Some inner membrane proteins are initially completely translocated into the matrixand are inserted into the inner membrane from the matrix side.
8. On the basis of their import characteristics, three different classes of IMS proteins canbe distinguished. Members of the first class contain bipartite presequences, membersof the second are imported by a folding trap mechanism, and members of the thirdassociate with binding sites on the outer or inner membrane, following translocationacross the TOM complex.
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FUTURE ISSUES
1. How are β-barrel proteins inserted into the outer membrane by the TOB/SAMcomplex? Do they assemble before, during, or after membrane insertion? Wheredoes the energy for the insertion reaction come from?
2. How are the TIM complexes gated? What is the specific role of presequences in thisprocess? Why is the membrane potential essential for gating?
3. How are membrane proteins inserted from the matrix into the inner membrane? Whatis the enzymatic activity of the Oxa1 complex? What are the molecular mechanismsthat are responsible for establishing the correct topology of membrane proteins?
4. How are proteins folded in the IMS? Is the folding and oxidation of thiol groupsmechanistically coupled in the IMS? Is there a protein with protein disulfide isomeraseactivity in the IMS?
ACKNOWLEDGMENTS
We apologize to authors whose work we were unable to cite owing to space limitations. Wethank Stephan Nussberger (University of Stuttgart, Germany) for the image used for Figure 2and Soledad Funes, Roman Kohl, Kai Hell, Dejana Mokranjac, and Doron Rapaport for com-ments on the manuscript.
LITERATURE CITED
1. Neupert W. 1997. Annu. Rev. Biochem. 66:863–9172. Karniely S, Regev-Rudzki N, Pines O. 2006. J. Mol. Biol. 358:396–4053. Regev-Rudzki N, Karniely S, Ben-Haim NN, Pines O. 2005. Mol. Biol. Cell 16:4163–714. Corral-Debrinski M, Blugeon C, Jacq C. 2000. Mol. Cell. Biol. 20:7881–925. Marc P, Margeot A, Devaux F, Blugeon C, Corral-Debrinski M, Jacq C. 2002. EMBO
Rep. 3:159–646. Mihara K, Omura T. 1996. Trends Cell Biol. 6:104–87. Young JC, Hoogenraad NJ, Hartl FU. 2003. Cell 112:41–508. Lister R, Murcha MW, Whelan J. 2003. Nucleic Acids Res. 31:325–279. Braun HP, Schmitz UK. 1997. Int. J. Biochem. Cell Biol. 29:1043–45
10. Gakh O, Cavadini P, Isaya G. 2002. Biochim. Biophys. Acta 1592:63–7711. Folsch H, Gaume B, Brunner M, Neupert W, Stuart RA. 1998. EMBO J. 17:6508–1512. Lee CM, Sedman J, Neupert W, Stuart RA. 1999. J. Biol. Chem. 274:20937–4213. Horie C, Suzuki H, Sakaguchi M, Mihara K. 2002. Mol. Biol. Cell 13:1615–2514. Horie C, Suzuki H, Sakaguchi M, Mihara K. 2003. J. Biol. Chem. 278:41462–7115. Habib SJ, Vasiljev A, Neupert W, Rapaport D. 2003. FEBS Lett. 555:511–1516. Kaufmann T, Schinzel A, Borner C. 2004. Trends Cell Biol. 14:8–1217. Hoogenraad NJ, Ward LA, Ryan MT. 2002. Biochim. Biophys. Acta 1592:97–10518. Braun HP, Schmitz UK. 1999. Planta 209:267–7419. Mori M, Terada K. 1998. Biochim. Biophys. Acta 1403:12–2720. Wu Y, Sha B. 2006. Nat. Struct. Mol. Biol. 13:589–9321. Chan NC, Likic VA, Waller RF, Mulhern TD, Lithgow T. 2006. J. Mol. Biol. 358:1010–
22
744 Neupert · Herrmann
Ann
u. R
ev. B
ioch
em. 2
007.
76:7
23-7
49. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by L
ouis
iana
Sta
te U
nive
rsity
- B
aton
Rou
ge o
n 11
/09/
07. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-30 ARI 8 May 2007 21:56
22. Abe Y, Shodai T, Muto T, Mihara K, Torii H, et al. 2000. Cell 100:551–6023. Yano M, Terada K, Mori M. 2003. J. Cell. Biol. 163:45–5624. van Wilpe S, Ryan MT, Hill K, Maarse AC, Meisinger C, et al. 1999. Nature 401:485–8925. Mayer A, Nargang FE, Neupert W, Lill R. 1995. EMBO J. 14:4204–1126. Hill K, Model K, Ryan MT, Dietmeier K, Martin F, et al. 1998. Nature 395:516–2127. Becker L, Bannwarth M, Meisinger C, Hill K, Model K, et al. 2005. J. Mol. Biol. 353:1011–
2028. Kunkele KP, Juin P, Pompa C, Nargang FE, Henry JP, et al. 1998. J. Biol. Chem.
273:31032–3929. Dekker PJ, Ryan MT, Brix J, Muller H, Honlinger A, Pfanner N. 1998. Mol. Cell. Biol.
18:6515–2430. Dietmeier K, Honlinger A, Bomer U, Dekker PJ, Eckerskorn C, et al. 1997. Nature
388:195–20031. Sherman EL, Go NE, Nargang FE. 2005. Mol. Biol. Cell 16:4172–8232. Kunkele KP, Heins S, Dembowski M, Nargang FE, Benz R, et al. 1998. Cell 93:1009–1933. Model K, Prinz T, Ruiz T, Radermacher M, Krimmer T, et al. 2002. J. Mol. Biol. 316:657–
6634. Ahting U, Thieffry M, Engelhardt H, Hegerl R, Neupert W, Nussberger S. 2001. J. Cell
Biol. 153:1151–6035. Ahting U, Thun C, Hegerl R, Typke D, Nargang FE, et al. 1999. J. Cell Biol. 147:959–6836. Esaki M, Shimizu H, Ono T, Yamamoto H, Kanamori T, et al. 2004. J. Biol. Chem.
279:45701–737. Bolliger L, Junne T, Schatz G, Lithgow T. 1995. EMBO J. 14:6318–2638. Mayer A, Neupert W, Lill R. 1995. Cell 80:127–3739. Komiya T, Rospert S, Koehler C, Looser R, Schatz G, Mihara K. 1998. EMBO J. 17:3886–
9840. Rapaport D, Kunkele KP, Dembowski M, Ahting U, Nargang F, et al. 1998. Mol. Cell.
Biol. 18:5256–6241. Yano M, Terada K, Mori M. 2004. J. Biol. Chem. 279:10808–1342. Esaki M, Kanamori T, Nishikawa S, Shin I, Schultz PG, Endo T. 2003. Nat. Struct. Biol.
10:988–9443. Chacinska A, Lind M, Frazier AE, Dudek J, Meisinger C, et al. 2005. Cell 120:817–2944. Donzeau M, Kaldi K, Adam A, Paschen S, Wanner G, et al. 2000. Cell 101:401–1245. Bauer MF, Sirrenberg C, Neupert W, Brunner M. 1996. Cell 87:33–4146. Geissler A, Chacinska A, Truscott KN, Wiedemann N, Brandner K, et al. 2002. Cell
111:507–1847. Yamamoto H, Esaki M, Kanamori T, Tamura Y, Nishikawa S, Endo T. 2002. Cell
111:519–2848. Truscott KN, Kovermann P, Geissler A, Merlin A, Meijer M, et al. 2001. Nat. Struct.
Biol. 8:1074–8249. Meier S, Neupert W, Herrmann JM. 2005. J. Biol. Chem. 280:7777–8550. Mokranjac D, Paschen SA, Kozany C, Prokisch H, Hoppins SC, et al. 2003. EMBO J.
22:816–2551. Meinecke M, Wagner R, Kovermann P, Guiard B, Mick DU, et al. 2006. Science 312:1523–
2652. Mokranjac D, Popov-Celeketic D, Hell K, Neupert W. 2005. J. Biol. Chem. 280:23437–4053. Berthold J, Bauer MF, Schneider HC, Klaus C, Dietmeier K, et al. 1995. Cell 81:1085–9354. Moro F, Sirrenberg C, Schneider HC, Neupert W, Brunner M. 1999. EMBO J. 18:3667–
75
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revi
ews.
org
by L
ouis
iana
Sta
te U
nive
rsity
- B
aton
Rou
ge o
n 11
/09/
07. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-30 ARI 8 May 2007 21:56
55. Josyula R, Jin Z, Fu Z, Sha B. 2006. J. Mol. Biol. 359:798–80456. Bukau B, Weissman J, Horwich A. 2006. Cell 125:443–5157. Young JC, Agashe VR, Siegers K, Hartl FU. 2004. Nat. Rev. Mol. Cell Biol. 5:781–9158. Schneider HC, Westermann B, Neupert W, Brunner M. 1996. EMBO J. 15:5796–80359. Liu Q, D’Silva P, Walter W, Marszalek J, Craig EA. 2003. Science 300:139–4160. Mokranjac D, Sichting M, Neupert W, Hell K. 2003. EMBO J. 22:4945–5661. Truscott KN, Voos W, Frazier AE, Lind M, Li Y, et al. 2003. J. Cell Biol. 163:707–1362. D’Silva PD, Schilke B, Walter W, Andrew A, Craig EA. 2003. Proc. Natl. Acad. Sci. USA
100:13839–4463. Kozany C, Mokranjac D, Sichting M, Neupert W, Hell K. 2004. Nat. Struct. Mol. Biol.
11:234–4164. Frazier AE, Dudek J, Guiard B, Voos W, Li Y, et al. 2004. Nat. Struct. Mol. Biol. 11:226–3365. D’Silva P, Liu QL, Walter W, Craig EA. 2004. Nat. Struct. Mol. Biol. 11:1084–9166. Li YF, Dudek J, Guiard B, Pfanner N, Rehling P, Voos W. 2004. J. Biol. Chem. 279:38047–
5467. D’Silva PR, Schilke B, Walter W, Craig EA. 2005. Proc. Natl. Acad. Sci. USA 102:12419–
2468. Mokranjac D, Bourenkov G, Hell K, Neupert W, Groll M. 2006. EMBO J. 25:4675–8569. van der Laan M, Chacinska A, Lind M, Perschil I, Sickmann A, et al. 2005. Mol. Cell. Biol.
25:7449–5870. Gallas MR, Dienhart MK, Stuart RA, Long RM. 2006. Mol. Biol. Cell 17:4051–6271. Tamura Y, Harada Y, Yamano K, Watanabe K, Ishikawa D, et al. 2006. J. Cell Biol.
174:631–3772. Matouschek A, Pfanner N, Voos W. 2000. EMBO Rep. 1:404–1073. Neupert W, Brunner M. 2002. Nat. Rev. Mol. Cell Biol. 3:555–6574. Ungermann C, Neupert W, Cyr DM. 1994. Science 266:1250–5375. Neupert W, Hartl FU, Craig EA, Pfanner N. 1990. Cell 63:447–5076. Schneider HC, Berthold J, Bauer MF, Dietmeier K, Guiard B, et al. 1994. Nature 371:768–
7477. Glick BS. 1995. Methods Enzymol. 260:224–3178. Horst M, Azem A, Schatz G, Glick BS. 1997. Biochim. Biophys. Acta 1318:71–7879. Okamoto K, Brinker A, Paschen SA, Moarefi I, Hayer-Hartl M, et al. 2002. EMBO J.
21:3659–7180. Huang S, Ratliff KS, Schwartz MP, Spenner JM, Matouschek A. 1999. Nat. Struct. Biol.
6:1132–3881. Wilcox AJ, Choy J, Bustamante C, Matouschek A. 2005. Proc. Natl. Acad. Sci. USA
102:15435–4082. Sato T, Esaki M, Fernandez JM, Endo T. 2005. Proc. Natl. Acad. Sci. USA 102:17999–800483. Rief M, Junker JP, Schlierf M, Hell K, Neupert W. 2006. Biophys. J. 91:2011–1284. Ainavarapu SR, Li L, Badilla CL, Fernandez JM. 2005. Biophys. J. 89:3337–4485. Ahting U, Waizenegger T, Neupert W, Rapaport D. 2005. J. Biol. Chem. 280:48–5386. Rapaport D. 2005. J. Cell Biol. 171:419–2387. Wimley WC. 2003. Curr. Opin. Struct. Biol. 13:404–1188. Paschen SA, Waizenegger T, Stan T, Preuss M, Cyrklaff M, et al. 2003. Nature 426:862–
6689. Wiedemann N, Kozjak V, Chacinska A, Schonfisch B, Rospert S, et al. 2003. Nature
424:565–7190. Kozjak V, Wiedemann N, Milenkovic D, Lohaus C, Meyer HE, et al. 2003. J. Biol. Chem.
278:48520–23
746 Neupert · Herrmann
Ann
u. R
ev. B
ioch
em. 2
007.
76:7
23-7
49. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by L
ouis
iana
Sta
te U
nive
rsity
- B
aton
Rou
ge o
n 11
/09/
07. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-30 ARI 8 May 2007 21:56
91. Gentle I, Gabriel K, Beech P, Waller R, Lithgow T. 2004. J. Cell Biol. 164:19–2492. Sanchez-Pulido L, Devos D, Genevrois S, Vicente M, Valencia A. 2003. Trends Biochem.
Sci. 28:523–2693. Voulhoux R, Bos MP, Geurtsen J, Mols M, Tommassen J. 2003. Science 299:262–6594. Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, Kahne D. 2005. Cell 121:235–4595. Ishikawa D, Yamamoto H, Tamura Y, Moritoh K, Endo T. 2004. J. Cell Biol. 166:621–2796. Milenkovic D, Kozjak V, Wiedemann N, Lohaus C, Meyer HE, et al. 2004. J. Biol. Chem.
279:22781–8597. Waizenegger T, Habib SJ, Lech M, Mokranjac D, Paschen SA, et al. 2004. EMBO Rep.
5:704–998. Abdul KM, Terada K, Yano M, Ryan MT, Streimann I, et al. 2000. Biochem. Biophys. Res.
Commun. 276:1028–3499. Glaser SM, Miller BR, Cumsky MG. 1990. Mol. Cell. Biol. 10:1873–81
100. van Loon APGM, Brandli AW, Schatz G. 1986. Cell 44:97–100101. Hartl FU, Schmidt B, Wachter E, Weiss H, Neupert W. 1986. Cell 47:939–51102. Hartl FU, Pfanner N, Neupert W. 1987. Biochem. Soc. Trans. 15:95–97103. Vergnolle MA, Baud C, Golovanov AP, Alcock F, Luciano P, et al. 2005. J. Mol. Biol.
351:839–49104. Webb CT, Gorman MA, Lazarou M, Ryan MT, Gulbis JM. 2006. Mol. Cell 21:123–33105. Lu H, Golovanov AP, Alcock F, Grossmann JG, Allen S, et al. 2004. J. Biol. Chem.
279:18959–66106. Korndorfer IP, Dommel MK, Skerra A. 2004. Nat. Struct. Mol. Biol. 11:1015–20107. Walton TA, Sousa MC. 2004. Mol. Cell 15:367–74108. Siegert R, Leroux MR, Scheufler C, Hartl FU, Moarefi I. 2000. Cell 103:621–32109. Habib SJ, Waizenegger T, Lech M, Neupert W, Rapaport D. 2005. J. Biol. Chem.
280:6434–40110. Wiedemann N, Truscott KN, Pfannschmidt S, Guiard B, Meisinger C, Pfanner N. 2004.
J. Biol. Chem. 279:18188–94111. Hoppins SC, Nargang FE. 2004. J. Biol. Chem. 279:12396–405112. Hofmann S, Rothbauer U, Muhlenbein N, Neupert W, Gerbitz KD, et al. 2002. J. Biol.
Chem. 277:23287–93113. Roesch K, Curran SP, Tranebjaerg L, Koehler CM. 2002. Hum. Mol. Genet. 11:477–86114. Allen S, Lu H, Thornton D, Tokatlidis K. 2003. J. Biol. Chem. 278:38505–13115. Curran SP, Leuenberger D, Oppliger W, Koehler CM. 2002. EMBO J. 21:942–53116. Curran SP, Leuenberger D, Schmidt E, Koehler CM. 2002. J. Cell Biol. 158:1017–27117. Sirrenberg C, Endres M, Folsch H, Stuart RA, Neupert W, Brunner M. 1998. Nature
391:912–15118. Lutz T, Neupert W, Herrmann JM. 2003. EMBO J. 22:4400–8119. Lu H, Woodburn J. 2005. J. Mol. Biol. 353:897–910120. Koehler CM. 2004. Trends Biochem. Sci. 29:1–4121. Curran SP, Leuenberger D, Leverich EP, Hwang DK, Beverly KN, Koehler CM. 2004.
J. Biol. Chem. 279:43744–51122. Sirrenberg C, Bauer MF, Guiard B, Neupert W, Brunner M. 1996. Nature 384:582–85123. Kovermann P, Truscott KN, Guiard B, Rehling P, Sepuri NB, et al. 2002. Mol. Cell
9:363–73124. Kerscher O, Holder J, Srinivasan M, Leung RS, Jensen RE. 1997. J. Cell Biol. 139:1663–75125. Kerscher O, Sepuri NB, Jensen RE. 2000. Mol. Biol. Cell 11:103–16126. Koehler CM, Murphy MP, Bally NA, Leuenberger D, Oppliger W, et al. 2000. Mol. Cell.
Biol. 20:1187–93
www.annualreviews.org • Translocation of Proteins into Mitochondria 747
Ann
u. R
ev. B
ioch
em. 2
007.
76:7
23-7
49. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by L
ouis
iana
Sta
te U
nive
rsity
- B
aton
Rou
ge o
n 11
/09/
07. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-30 ARI 8 May 2007 21:56
127. Endres M, Neupert W, Brunner M. 1999. EMBO J. 18:3214–21128. Wiedemann N, Pfanner N, Ryan MT. 2001. EMBO J. 20:951–60129. Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trezeguet V, Lauquin GJ, Brandolin
G. 2003. Nature 426:39–44130. Brix J, Rudiger S, Bukau B, Schneider-Mergener J, Pfanner N. 1999. J. Biol. Chem.
274:16522–30131. Brix J, Ziegler GA, Dietmeier K, Schneider-Mergener J, Schulz GE, Pfanner N. 2000.
J. Mol. Biol. 303:479–88132. Vasiljev A, Ahting U, Nargang FE, Go NE, Habib SJ, et al. 2004. Mol. Biol. Cell 15:1445–
58133. Brandner K, Rehling P, Truscott KN. 2005. J. Biol. Chem. 280:6215–21134. Pfanner N, Neupert W. 1987. J. Biol. Chem. 262:7528–36135. Komiya T, Rospert S, Schatz G, Mihara K. 1997. EMBO J. 16:4267–75136. Ryan MT, Muller H, Pfanner N. 1999. J. Biol. Chem. 274:20619–27137. Truscott KN, Wiedemann N, Rehling P, Muller H, Meisinger C, et al. 2002. Mol. Cell.
Biol. 22:7780–89138. Koehler CM, Merchant S, Oppliger W, Schmid K, Jarosche E, et al. 1998. EMBO J.
17:6477–86139. Webb CT, Gorman MA, Lazarou M, Ryan MT, Gulbis JM. 2006. Mol. Cell 21:123–33140. Panneels V, Schussler U, Costagliola S, Sinning I. 2003. Biochem. Biophys. Res. Commun.
300:65–74141. Dyall SD, Agius SC, De Marcos Lousa C, Trezeguet V, Tokatlidis K. 2003. J. Biol. Chem.
278:26757–64142. Kaldi K, Bauer MF, Sirrenberg C, Neupert W, Brunner M. 1998. EMBO J. 17:1569–76143. Kurz M, Martin H, Rassow J, Pfanner N, Ryan MT. 1999. Mol. Biol. Cell 10:2461–74144. Leuenberger D, Bally NA, Schatz G, Koehler CM. 1999. EMBO J. 18:4816–22145. Davis AJ, Sepuri NB, Holder J, Johnson AE, Jensen RE. 2000. J. Cell Biol. 150:1271–82146. Paschen SA, Rothbauer U, Kaldi K, Bauer MF, Neupert W, Brunner M. 2000. EMBO
J. 19:6392–400147. Tranebjaerg L, Schwartz C, Eriksen H, Andreasson S, Ponjavic V, et al. 1995. J. Med.
Genet. 32:257–63148. Jin H, May M, Tranebjaerg L, Kendall E, Fontan G, et al. 1996. Nat. Genet. 14:177–80149. Rothbauer U, Hofmann S, Muhlenbein N, Paschen SA, Gerbitz KD, et al. 2001. J. Biol.
Chem. 276:37327–34150. Roesch K, Hynds PJ, Varga R, Tranebjaerg L, Koehler CM. 2004. Hum. Mol. Genet.
13:2101–11151. Gartner F, Voos W, Querol A, Miller BR, Craig EA, et al. 1995. J. Biol. Chem. 270:3788–
95152. Miller BR, Cumsky MG. 1993. J. Cell Biol. 121:1021–29153. Meier S, Neupert W, Herrmann JM. 2005. J. Cell Biol. 170:881–88154. Rojo EE, Guiard B, Neupert W, Stuart RA. 1998. J. Biol. Chem. 273:8040–47155. Folsch H, Guiard B, Neupert W, Stuart RA. 1996. EMBO J. 15:479–87156. Rojo EE, Stuart RA, Neupert W. 1995. EMBO J. 14:3445–51157. Herrmann JM, Neupert W, Stuart RA. 1997. EMBO J. 16:2217–26158. Baumann F, Neupert W, Herrmann JM. 2002. J. Biol. Chem. 277:21405–13159. Funes S, Nargang FE, Neupert W, Herrmann JM. 2004. Mol. Biol. Cell 15:1853–61160. Herrmann JM, Koll H, Cook RA, Neupert W, Stuart RA. 1995. J. Biol. Chem. 270:27079–
86
748 Neupert · Herrmann
Ann
u. R
ev. B
ioch
em. 2
007.
76:7
23-7
49. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by L
ouis
iana
Sta
te U
nive
rsity
- B
aton
Rou
ge o
n 11
/09/
07. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-30 ARI 8 May 2007 21:56
161. Hell K, Herrmann J, Pratje E, Neupert W, Stuart RA. 1997. FEBS Lett. 418:367–70162. Hell K, Herrmann JM, Pratje E, Neupert W, Stuart RA. 1998. Proc. Natl. Acad. Sci. USA
95:2250–55163. Stuart RA. 2002. Biochem. Biophys. Acta 1592:79–87164. Herrmann JM, Neupert W. 2003. IUBMB Life 55:219–25165. Nett JH, Trumpower BL. 1996. J. Biol. Chem. 271:26713–16166. Golik P, Bonnefoy N, Szczepanek T, Saint-Georges Y, Lazowska J. 2003. Proc. Natl.
Acad. Sci. USA 100:8844–49167. Burri L, Strahm Y, Hawkins CJ, Gentle IE, Puryer MA, et al. 2005. Mol. Biol. Cell
16:2926–33168. Nunnari J, Fox D, Walter P. 1993. Science 262:1997–2004169. Esser K, Tursun B, Ingenhoven M, Michaelis G, Pratje E. 2002. J. Mol. Biol. 323:835–43170. McQuibban GA, Saurya S, Freeman M. 2003. Nature 423:537–41171. Herlan M, Vogel F, Bornhovd C, Neupert W, Reichert AS. 2003. J. Biol. Chem.
278:27781–88172. Langer T. 2000. Trends Biochem. Sci. 25:247–51173. Herlan M, Bornhovd C, Hell K, Neupert W, Reichert AS. 2004. J. Cell Biol. 165:167–73174. Diekert K, de Kroon AI, Ahting U, Niggemeyer B, Neupert W, et al. 2001. EMBO J.
20:5626–35175. Dumont ME, Ernst JF, Sherman F. 1988. J. Biol. Chem. 263:15928–37176. Nargang FE, Drygas ME, Kwong PL, Nicholson DW, Neupert W. 1988. J. Biol. Chem.
263:9388–94177. Chacinska A, Pfannschmidt S, Wiedemann N, Kozjak V, Szklarz LKS, et al. 2004. EMBO
J. 23:3735–46178. Terziyska N, Lutz T, Kozany C, Mokranjac D, Mesecke N, et al. 2004. FEBS Lett.
579:179–84179. Naoe M, Ohwa Y, Ishikawa D, Ohshima C, Nishikawa S, et al. 2004. J. Biol. Chem.
279:47815–21180. Mesecke N, Terziyska N, Kozany C, Baumann F, Neupert W, et al. 2005. Cell 121:1059–
69181. Allen S, Balabanidou V, Sideris DP, Lisowsky T, Tokatlidis K. 2005. J. Mol. Biol. 353:937–
44182. Rissler M, Wiedemann N, Pfannschmidt S, Gabriel K, Guiard B, et al. 2005. J. Mol. Biol.
353:485–92183. Farrell SR, Thorpe C. 2005. Biochemistry 44:1532–41184. Field LS, Furukawa Y, O’Halloran TV, Culotta VC. 2003. J. Biol. Chem. 278:28052–59185. Emanuelsson O, Nielsen H, Brunak S, von Heijne G. 2000. J. Mol. Biol. 300:1005–16186. Nakai K, Horton P. 1999. Trends Biochem. Sci. 24:34–36187. Guda C, Fahy E, Subramaniam S. 2004. Bioinformatics 20:1785–94188. Claros MG. 1995. Comput. Appl. Biosci. 11:441–47189. Small I, Peeters N, Legeai F, Lurin C. 2004. Proteomics 4:1581–90
RELATED RESOURCES
Falkenberg M, Larsson N-G, Gustafsson C. 2007. Annu. Rev. Biochem. 76:679–99Hoppins S, Lackner L, Nunnari J. 2007. Annu. Rev. Biochem. 76:751–80Ryan MT, Hoogenraad NJ. 2007. Annu. Rev. Biochem. 76:701–22Schatz G. 2007. Annu. Rev. Biochem. 76:673–78Elofsson A, von Heijne G. 2007. Annu. Rev. Biochem. 76:125–40Wallace DC. 2007. Annu. Rev. Biochem. 76:781–821
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Annual Review ofBiochemistry
Volume 76, 2007Contents
Mitochondrial Theme
The Magic GardenGottfried Schatz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �673
DNA Replication and Transcription in Mammalian MitochondriaMaria Falkenberg, Nils-Göran Larsson, and Claes M. Gustafsson � � � � � � � � � � � � � � � � � � �679
Mitochondrial-Nuclear CommunicationsMichael T. Ryan and Nicholas J. Hoogenraad � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �701
Translocation of Proteins into MitochondriaWalter Neupert and Johannes M. Herrmann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �723
The Machines that Divide and Fuse MitochondriaSuzanne Hoppins, Laura Lackner, and Jodi Nunnari � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �751
Why Do We Still Have a Maternally Inherited Mitochondrial DNA?Insights from Evolutionary MedicineDouglas C. Wallace � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �781
Molecular Mechanisms of Antibody Somatic HypermutationJavier M. Di Noia and Michael S. Neuberger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1
Structure and Mechanism of Helicases and Nucleic Acid TranslocasesMartin R. Singleton, Mark S. Dillingham, and Dale B. Wigley � � � � � � � � � � � � � � � � � � � � � � 23
The Nonsense-Mediated Decay RNA Surveillance PathwayYao-Fu Chang, J. Saadi Imam, Miles F. Wilkinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 51
Functions of Site-Specific Histone Acetylation and DeacetylationMona D. Shahbazian and Michael Grunstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 75
The tmRNA System for Translational Surveillance and Ribosome RescueSean D. Moore and Robert T. Sauer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �101
Membrane Protein Structure: Prediction versus RealityArne Elofsson and Gunnar von Heijne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �125
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Structure and Function of Toll Receptors and Their LigandsNicholas J. Gay and Monique Gangloff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �141
The Role of Mass Spectrometry in Structure Elucidation of DynamicProtein ComplexesMichal Sharon and Carol V. Robinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �167
Structure and Mechanism of the 6-Deoxyerythronolide B SynthaseChaitan Khosla, Yinyan Tang, Alice Y. Chen, Nathan A. Schnarr,
and David E. Cane � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �195
The Biochemistry of Methane OxidationAmanda S. Hakemian and Amy C. Rosenzweig � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �223
Anthrax Toxin: Receptor Binding, Internalization, Pore Formation,and TranslocationJohn A.T. Young and R. John Collier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �243
Synapses: Sites of Cell Recognition, Adhesion, and FunctionalSpecificationSoichiro Yamada and W. James Nelson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �267
Lipid A Modification Systems in Gram-negative BacteriaChristian R.H. Raetz, C. Michael Reynolds, M. Stephen Trent,
and Russell E. Bishop � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �295
Chemical Evolution as a Tool for Molecular DiscoveryS. Jarrett Wrenn and Pehr B. Harbury � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �331
Molecular Mechanisms of Magnetosome FormationArash Komeili � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �351
Modulation of the Ryanodine Receptor and Intracellular CalciumRan Zalk, Stephan E. Lehnart, and Andrew R. Marks � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �367
TRP ChannelsKartik Venkatachalam and Craig Montell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �387
Studying Individual Events in BiologyStefan Wennmalm and Sanford M. Simon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �419
Signaling Pathways Downstream of Pattern-Recognition Receptorsand Their Cross TalkMyeong Sup Lee and Young-Joon Kim � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �447
Biochemistry and Physiology of Cyclic Nucleotide Phosphodiesterases:Essential Components in Cyclic Nucleotide SignalingMarco Conti and Joseph Beavo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �481
The Eyes Absent Family of Phosphotyrosine Phosphatases: Propertiesand Roles in Developmental Regulation of TranscriptionJennifer Jemc and Ilaria Rebay � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �513
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Assembly Dynamics of the Bacterial MinCDE System and SpatialRegulation of the Z RingJoe Lutkenhaus � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �539
Structures and Functions of Yeast Kinetochore ComplexesStefan Westermann, David G. Drubin, and Georjana Barnes � � � � � � � � � � � � � � � � � � � � � � � �563
Mechanism and Function of Formins in the Control of Actin AssemblyBruce L. Goode and Michael J. Eck � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �593
Unsolved Mysteries in Membrane TrafficSuzanne R. Pfeffer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �629
Structural Biology of Nucleocytoplasmic TransportAtlanta Cook, Fulvia Bono, Martin Jinek, and Elena Conti � � � � � � � � � � � � � � � � � � � � � � � � � �647
The Postsynaptic Architecture of Excitatory Synapses: A MoreQuantitative ViewMorgan Sheng and Casper C. Hoogenraad � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �823
Indexes
Cumulative Index of Contributing Authors, Volumes 72–76 � � � � � � � � � � � � � � � � � � � � � � � �849
Cumulative Index of Chapter Titles, Volumes 72–76 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �853
Errata
An online log of corrections to Annual Review of Biochemistry chapters (if any, 1997to the present) may be found at http://biochem.annualreviews.org/errata.shtml
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