Critical Review

Mitochondrial Protein Homeostasis Aksana Varabyova1

Diana Stojanovski2

Agnieszka Chacinska1*

1International Institute of Molecular and Cell Biology, 02-109 Warsaw,Poland2Department of Biochemistry and Molecular Biology, Bio21 MolecularScience and Biotechnology Institute, The University of Melbourne,Parkville, 3010, VIC, Australia

Abstract

Mitochondria use 800–1,500 proteins to perform their biological

functions in the eukaryotic cells. Distinct transport and sorting

mechanisms are responsible for the delivery of proteins to the

correct location within mitochondria. Mitochondrial proteins

undergo processing events and form functional assemblies.

Finally, non-functional proteins are cleared to maintain healthy

mitochondria. We provide an overview of the processes collec-

tively contributing to the maintenance of mitochondrial protein

homeostasis, which is critical for cell physiology and survival.

VC 2013 IUBMB Life, 00(00):000000, 2013.

Keywords: mitochondria; protein assembly; protein degradation;

protein import; protein processing

Introduction

Proteins are the executors of all cellular processes. Thus, pro-duction of proteins as well as maintaining protein homeostasisis a major challenge for the cell. To perform these essentialtasks cells have developed elaborate mechanisms that regulatethe birth, maturation, and eventual death of a protein. In thehighly compartmentalized eukaryotic cell, the majority ofproteins do not function in their place of synthesis within thecytosol, but rather must be trafficked to one of the variousmembrane enclosed compartments. One such compartmentthat is critical for cellular function and survival is the mito-chondrion. The biogenesis of mitochondrial proteins isextremely challenging due to organelles architecture, whichconsists of the two membrane barriers, the outer and innermitochondrial membranes that create the borders for twoaqueous compartments, the mitochondrial intermembranespace (IMS), and matrix. Although mitochondria possess theirown genome, it encodes only a handful of mitochondrialproteins. The large majority of mitochondrial proteins come

from the cytosol, where they are synthesized based on geneticinformation encoded by the nuclear DNA (1–3). Following theirsynthesis, mitochondrial precursor proteins are subjected to avariety of processes that lead to their targeting to the organ-elle and subsequent sorting to the appropriate mitochondrialsub-compartment. Next, proteins mature via various process-ing events and assemble into functional complexes. Finally,once they have carried out their purpose or in response todamage, mitochondrial proteins have to be efficiently removed.In this review, we summarize the developments that haveshaped our current understanding of the processes thatcontribute to the maintenance of mitochondrial proteinhomeostasis.

Influx of Proteins into Mitochondria

The majority of mitochondrial proteins enter the organelle in apost-translational manner (Fig. 1). The entry of virtually allmitochondrial precursor proteins is facilitated by the Translo-case of the Outer mitochondrial Membrane (TOM complex)(4–7). The central subunit of the TOM complex is Tom40, ab-barrel and channel forming protein that allows precursorproteins to be transported from the cytosol into the mitochon-dria. This transport of precursors is assisted by several otherTom subunits that regulate the architecture and function ofthe TOM translocase. These components can be divided intotwo groups: (i) the core TOM subunits, which include thecentral receptor, Tom22 and the small Tom proteins, which

*Address for correspondence to: Agnieszka Chacinska, InternationalInstitute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw,Poland. Tel: þ48-22-5970779. Fax: þ48-22-5970715. E-mail: achacinska@iimcb.gov.pl.Received November 2012; accepted 29 November 2012DOI: 10.1002/iub.1122Published online in Wiley Online Library(wileyonlinelibrary.com)

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regulate complex dynamics; and (ii) the peripheral receptorsubunits, Tom70 and Tom20, which are more loosely associ-ated with the complex. Following synthesis and their journeyto mitochondria, precursor proteins initially encounter theTom70 and Tom20 receptors. Tom70 and Tom20 recognizedifferent features and targeting signals within the precursorsand this initiates entry into a defined biogenesis pathway. Fur-thermore, a series of binding sites with increasing affinitiesprovided by the domains of TOM receptors, Tom22 and Tom5,drive translocation of the precursor through the TOM channel.Thus, the recognition of specific targeting elements and coop-eration between the TOM subunits allows unidirectional trans-port into the TOM channel. Interestingly, new avenues ofresearch have identified phosphorylation as an importantfactor in the regulation of the TOM complex. Protein kinase A(PKA) phosphorylates Tom70 leading to inhibition of the recep-tor and ultimately impaired delivery of Tom70-dependent sub-strates into mitochondria. Conversely, the assembly of theTOM complex itself is positively regulated by the action ofcasein kinase 2 (CK2), by the direct action of this kinase on thecentral receptor, Tom22 (8,9). These examples demonstratethat mitochondrial translocases are targets of intracellularsignaling and that the transport of mitochondrial proteins isintegrated into the regulatory circuits that determine the fate

and physiology of the cell. It will be interesting to see whetherthe other mitochondrial translocation machineries alsoundergo similar regulations.

The early events that precede TOM translocation are lessdefined. Mitochondrial precursors are transient residents ofthe cytosol, where they are synthesized. However, to maintainimport competence these precursors need to be kept in anunfolded or partially unfolded conformation by the cytosolicchaperones (Fig. 1). The classical example of precursors thatrequire cytosolic guidance is provided by inner membranemetabolite carriers, such as the ADP/ATP carrier. Theselargely hydrophobic proteins require the action of Hsp70 andHsp90 to shield their hydrophobic patches and facilitate theirpassage to the Tom70 receptor in the ATP-dependent manner(10,11).

Under normal conditions, the passage of mitochondrialprecursors through the cytosol is rapid. Moreover, there areindications that the delivery of proteins to mitochondria maybe facilitated in a co-translational manner (Fig. 1). Thekinetics of import into mitochondria is likely to be influencedby the localization of translating ribosomes loaded with thespecific mRNA molecule. Pioneering work revealed the pres-ence of cytosolic ribosomes on the surface of mitochondria(12,13). Furthermore, mRNAs coding for a large group of

Synthesis of mitochondrial precursor proteins in the cytosol and their import into mitochondria. Mitochondrial precursors syn-

thesized on cytosolic ribosome are imported into mitochondria in a post-translational manner. Chaperones assist in delivery of

the newly synthesized proteins to the main entry gate of mitochondria, the TOM complex. During co-translational import, the

nascent polypeptide chain is directly transferred from ribosome to the TOM complex. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]

FIG 1

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mitochondrial precursors are found in close proximity to mito-chondria and a role for the Tom20 receptor as well as themRNA-binding protein Puf3 have been established in the pro-cess of mRNA localization to mitochondria (14–18). Anextreme example of complete dependence on active translationfor mitochondrial import has been reported for fumarase (19)and Sod2 (20). Interestingly, the ribosome-associated NACcomplex (Nascent chain-Associated Complex) has been identi-fied as a component that increases the rate of protein translo-cation into mitochondria (19,21). The possibility of a directinterplay between cytosolic ribosomes and mitochondrialtranslocases is further supported by studies that haveemployed mitochondrial precursors expressed from mRNAlacking stop codons (‘‘nonstop’’ mRNA). Nonstop mRNAs andprotein nascent chains remain stalled on ribosomes and repre-sent a danger for the cell. The recent study demonstrated thatnon-stop mitochondrial proteins in addition to blocking ribo-somes are also engaged with the TOM translocase. Clearance

factors associated with the ribosomes operate at the level ofRNA and proteins to prevent such situations (22,23). It istempting to hypothesize the existence of mechanisms that con-trol spatiotemporal coordination of protein synthesis andtransport is important for maintaining healthy mitochondria.

Pathways for Protein Sorting in theMitochondria

The targeting signals found within a mitochondrial precursordefine the biogenesis pathway and components that areinvolved at TOM and post-TOM association (Fig. 2). The classi-cal import pathway into mitochondria relies on a well-definedtargeting signal called a presequence (4,5,24,25). This posi-tively charged amphipathic helix is located at the N-terminusof a large fraction of mitochondrial proteins, and is cleaved offduring the maturation process (discussed later). After passage

Different pathways for protein sorting in mitochondria. Mitochondrial precursor proteins are transferred across the outer mito-

chondrial membrane via the TOM complex. Import of precursor proteins with typical cysteine residues arrangements (CXnC) is

coupled to their oxidative folding. Precursor proteins containing a positively charged presequence are directed to the TIM23

complex, which is responsible for their translocation into or across the inner mitochondrial membrane in a membrane potential

(Dw)-dependent manner. Hydrophobic membrane proteins are guided by small Tim proteins to the TIM22 complex for inner

membrane integration, in the case of b-barrel precursor proteins, to the SAM complex in the outer membrane of mitochondria.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIG 2

Varabyova et al. 3

across the outer mitochondrial membrane presequence-containing precursors are recognized by the inner membranepresequence translocase, the TIM23 complex (Fig. 2). The firstsubunit of the TIM23 complex encountered by an incomingprecursor protein on the trans-side of the TOM complex isTim50 (26–29). Tim50 passes the presequence to the channelforming unit of TIM23 formed by the Tim23 and Tim17 pro-teins. On route from TOM to the TIM23 complex the electro-chemical potential across the mitochondrial inner membraneserves as a driving force. The membrane potential drives theprecursor proteins with their positively charged presequenceacross the TIM23 translocase to the mitochondrial matrix. Thecomplete passage of precursors into the mitochondrial matrixis achieved via the action of the presequence translocase-asso-ciated motor complex (PAM complex). PAM has mitochondrialHsp70 as its central player and through successive rounds ofATP binding and hydrolysis Hsp70 modulates translocation ofprecursors across the inner membrane (4,5,24,25).

The TIM23 translocase is also involved in the biogenesis ofmembrane proteins that carry a presequence and at least onetransmembrane segment. The early biogenesis steps of thisclass of proteins are similar to those of the classical substratesdescribed above. However, once translocation through theTIM23 complex is initiated the pathways start to diverge. Thetransmembrane domain, which is positioned downstream ofthe presequence, serves as a ‘‘stop-transfer’’ signal and haltstranslocation of the precursor in the TIM23 channel. This pro-cess can be performed by TIM23 that lacks the associatedPAM complex and is often referred to as TIM23SORT. TIM23-SORT is distinguished by the presence of additional subunit,Tim21 and this form of the complex is competent for proteinintegration into the inner membrane (30–33). Furthermore,Tim21 has been shown to connect the TIM23 complex to therespiratory chain complexes III and IV (34). Thus, the interplayof signals within precursors decoded by the TIM23 translocasedrives protein translocation across or into the mitochondrialinner membrane.

The majority of mitochondrial proteins that reside withinthe outer membrane, IMS, and inner membrane do not pos-sess a classical cleavable presequence. Popular examplesinclude the carrier proteins of the inner membrane, b-barrelproteins of the outer membrane, and soluble cysteine rich pro-teins of the IMS. The large family of inner membrane proteinsknown as the metabolite carriers, such as the ADP/ATP carrier(AAC), function in the transfer of metabolites between the mi-tochondrial matrix and IMS. The carrier proteins preferentiallybind to the Tom70 receptor and to complete the transferthrough TOM and across the outer membrane, the carrier pro-teins are recognized by chaperone complexes within the IMSbelonging to the small Tim family (4,24). The small Tim chap-erones assist the hydrophobic precursors in the passagethrough the IMS and subsequent docking onto the inner mem-brane carrier translocase (TIM22 complex), which is the ma-chinery dedicated to the biogenesis of this class of proteins(Fig. 2). The TIM22 complex is composed of the channel form-

ing subunit, Tim22 and the auxiliary subunits, Tim54, Tim18,and Sdh3, which are important for the efficient binding of pre-cursor proteins to TIM22 and for the assembly of the TIM22translocase (4,24,35,36). The import of carrier proteins intothe inner membrane depends on the electrochemical potentialacross the inner membrane.

The small Tim chaperones also take part in the transportpathway of b-barrel proteins, such as porins and Tom40,which are exclusively found in the outer membrane of mito-chondria (4,7,37). The b-barrel proteins do not enter the outermitochondrial membrane from the cytosolic side but ratherare transferred via TOM into the IMS and are subsequentlydirected to the outer membrane with help of the small Timproteins (Fig. 2). The small Tim proteins chaperone the b-bar-rel proteins to the Sorting and Assembly Machinery (SAM com-plex) of the outer membrane (38,39). The SAM complex recog-nizes its substrate by virtue of a specific b-signal found at theC-terminus of b-barrel proteins and mediates their integrationinto the outer membrane (40). Interestingly, SAM is alsoresponsible for early assembly events of b-barrel containingcomplexes such as the TOM complex and this task is achievedby the cooperation of the SAM complex with the multifunc-tional proteins such as Mdm10 and Mdm12, which are addi-tionally responsible for maintaining mitochondrial shape anddirect contacts with the endoplasmic reticulum (41–43).

Many IMS proteins have characteristic structural features,including the presence of conserved cysteine motifs. These cys-teines residues are reduced in the cytosol, however once theprecursor is imported into mitochondria they are rapidly oxi-dized to disulfide bonds by the Mitochondrial IMS Import andAssembly Machinery, MIA (Fig. 2) (44–46). This process isfacilitated by two essential components of the MIA pathway,Mia40 and Erv1 and leads to trapping of IMS precursorswithin the organelle due to oxidative folding (47–49). Mecha-nistic studies have demonstrated that the MIA pathway relieson the coordinated action of its two central players, Mia40 andErv1, and also on the reducing power of glutathione, to exe-cute disulfide transfer reactions efficiently by minimizingunproductive intermediate stages (50–52). These featuresensure that the MIA system works efficiently despite the factthat the IMS is continuous with the reducing environment ofthe cytosol (53). Mia40 recognizes its substrate by the presenceof a hydrophobic cysteine-containing signal called MISS/ITS assoon as the precursor emerges from the TOM complex (54–56). However, Mia40 is anchored in the inner membrane,which is folded into cristae, and it remained unclear how IMSprecursors reached Mia40 for their rapid oxidation. Recentwork has provided insight into this question by the identifica-tion of Fcj1 (Mitofilin in higher Eukaryotes) as a factor respon-sible for recruiting Mia40 to the vicinity of TOM (56). Theinteractions of Fcj1, Mia40, and TOM components brings tolight an interesting regulatory mechanism where a stimulatoryeffect is achieved by the accurate positioning of Mia40 in thevicinity of TOM thereby positively influencing the activity of theMIA pathway. This phenomenon is likely to be more universal

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4 Mitochondrial Protein Homeostasis

as Fcj1 is a core component of the MINOS/MitOS/MICOS com-plex responsible for maintaining the unique inner membranearchitecture and it interacts with multiple other proteincomplexes in the inner and outer mitochondrial membrane(56–59).

In summary, the signals within mitochondrial precursorproteins are decoded by specific translocases and this deter-mines the localization of the protein inside mitochondria(Fig. 2). The mitochondrial translocases are multifacetedmolecular machines and perform various functions including:(i) precursor recognition (receptor), (ii) precursor translocationacross membranes (channel), (iii) precursor integration intomembranes (integrase), and (iv) prevention of precursor’sunwanted and hazardous interactions (chaperone-like). Thesefeatures all ensure that the mitochondrial protein content andorganelle integrity is upheld.

Specific Transport Pathwaysand Their Interplays withMajor Translocases

In addition to mechanisms that operate in delivery of proteinsto the four major pathways (TIM23, TIM22, SAM, and MIA),there is a growing evidence for the presence of specializedpathways that have developed to meet the needs of a smallsubset or even single proteins. The outer membrane providesa great example of specialized import and assembly pathways(7,37). In addition to b-barrel proteins, the outer membraneharbors a variety of proteins that are anchored via one ormore a-helical transmembrane segments. In contrast to theb-barrel protein assembly pathway, which involved the trans-fer of a precursor to the IMS side of the outer mitochondrialmembrane, these proteins are inserted into outer membranedirectly from the cytosol. How the TOM complex mediates theinsertion of outer membrane proteins with a-helical trans-membrane domains remains enigmatic. Use of a pore formedby a single Tom40 molecule would require the energeticallyunfavorable breaking of the b-barrel for release of the trans-membrane domains into the membrane. A more plausiblemodel is that a ‘‘pore’’ formed between several Tom40 subu-nits could open laterally through the rearrangement of thesesubunits permitting membrane insertion of proteins. Indeed,this mechanism was suggested for fusion proteins arrested atthe level of the TOM complex (60). Other possibilities mayinclude a complete bypass of the TOM complex or use theinterface between the TOM complex and the lipid bilayer as ameans to integrate into the membrane.

Insertion of N-terminally anchored proteins, such asTom20 and Tom70, is facilitated by the outer membraneinsertase Mim1 (61–63). It remains unknown whether a pro-tein-based machinery exists for the insertion of C-terminallyanchored (tail-anchored) proteins into the outer membrane. Itappears that the lipid composition of the outer membrane is a

major requirement for integrating of the tail-anchored pro-teins (64–66). Interestingly, in the case of Tom22, the receptorand organizer of TOM that is also C-terminally anchored, arequirement for both the TOM and SAM complexes for its bio-genesis has been demonstrated (67). Tail-anchored proteinsare present in various cellular membranes, and must first betargeted to the endoplasmic reticulum before reaching theirfinal destinations via vesicular trafficking. Transport from thecytosolic ribosomes into the endoplasmic reticulum (ER) mem-brane is assisted by the Golgi to ER Traffic (GET) machinery(68). GET has not been demonstrated so far to influence thebiogenesis of the mitochondrial outer membrane proteins. Thisraises the question of how the GET machinery differentiatesbetween the tail-anchored proteins targeted to the ER and mi-tochondria. In summary, protein insertion pathways into theouter mitochondrial membrane display remarkable mechanis-tic variety, which are not yet completely understood. Giventhat outer membrane proteins play important roles in a pleth-ora of processes, including mitochondrial dynamics, mitoph-agy, protein biogenesis, and regulation of the programmed celldeath, this area of research is of high interest.

Discrete import pathways have been described for IMSsubstrates, including mitochondrial heme lyases that aretrapped in the IMS in a process that involves folding and high-affinity interactions (69). More recently, the IMS residentMdm35 was shown to serve as a folding and assembly trap forthe Ups proteins, Ups1 and Ups2, which function in lipid trans-fer (70). These two examples support the folding trap modelfor IMS proteins based on retention of proteins within the IMSthrough folding. This prevents back-sliding of proteins into thecytosol and ultimately determines the directionality of proteintransport.

Proteins encoded by the mitochondrial genome are synthe-sized on mitochondrial ribosomes and also use interestingmechanisms for their biogenesis. This group of proteins consti-tutes less than 1% of the mitochondrial proteome, but is ofgreat functional importance for the inner membrane-embed-ded respiratory chain complexes involved in oxidative phos-phorylation. The process by which these proteins are inte-grated into the inner membrane is referred to as ‘‘proteinexport’’ as it relies on the proteins being trafficked out of thematrix to the inner membrane. A number of proteins governthe inner-membrane localized translation and assist in themembrane insertion of mitochondrially synthesized polypep-tides (71–73). Oxa1 is the best characterized export componentconserved among prokaryotic and eukaryotic organisms. Thischannel forming factor is in direct contact with the translatingribosome (74,75). Remarkably, Oxa1 is also involved in theexport of inner membrane proteins that use the TIM23 com-plex for their partial translocation into the matrix. An interest-ing case has been described for the multispanning inner mem-brane ABC transporter, Mdl1 (76). This protein contains threedomains, each centered around one pair of transmembranedomains. The first and third domains are laterally sorted tothe inner membrane by the TIM23 complex. In contrast, the

Varabyova et al. 5

middle domain is translocated into the matrix by TIM23 in anHsp70-dependent manner and is subsequently subjected toexport into the inner membrane with the help of Oxa1. Thus,Mdl1 provides an interesting example of protein with a compli-cated domain architecture, which utilizes two alternativemechanisms to adopt correct topology in the inner mitochon-drial membrane. This involves the cooperative action of theTIM23 translocase and the Oxa1 export machinery (76).

Another example of protein export from the matrix intothe inner membrane is the pathway for Rieske protein, acomponent of complex III of the respiratory chain. TheRieske protein is first transported to the mitochondrialmatrix and binds a cofactor, iron-sulfur cluster, whichallows the protein to adopt a partially folded conformation.In this form, the Rieske protein is transported to the innermembrane by the Bcs1 protein, a member of highly con-served AAAþ family with various functions in the cell (77).This extreme example of a ‘‘private’’ translocation pathwayraises the possibility that many more specific pathways existin mitochondria.

Maturation of Mitochondrial Proteins

Once protein import and sorting have been achieved the mito-chondrial precursor is then left to undergo maturation and inmany cases assemble into a functional complex. For a largenumber of TIM23 substrates that enter the mitochondrialmatrix and inner membrane, this involves removal of the pre-sequence (Fig. 3) (78,79). Cleavage of the N-terminal prese-quence is facilitated by the mitochondrial membrane protease,MPP. This cleavage is not functionally coupled to proteintranslocation, although it frequently appears immediately aftera presequence emerges on the matrix side. Interestingly, twoadditional proteases, Icp55 and Oct1, were identified to play arole in further processing steps following MPP cleavage for adefined subset of proteins (Fig. 3) (80,81). The N-terminalamino acid residue of a protein contributes to the stabilityof that protein in the eukaryotic cytosol and bacteria, andthis well grounded phenomenon is called the N-end rule (82).It appeared that MPP cleavage in some cases exposes a desta-bilizing amino acid residue at the N-termini of some

Proteolytic processing of mitochondrial proteins. The N-terminal positively charged presequence drives precursor proteins

through the TIM23 translocase into the matrix, where it is removed by mitochondrial processing peptidase, MPP. In some case,

the Icp55 or Oct1 proteases catalyze a further cleavage of single amino acid residue or an octapeptide from the N-terminus of

the precursor. In some cases, the presequence is followed by a hydrophobic signal and in this case the precursor protein is lat-

erally released into the inner membrane and the hydrophobic signal is removed by the inner membrane peptidase, IMP. Addi-

tionally, other proteases including the rhomboid protease (Pcp1), m-AAA, and Atp23 have been reported to execute specific

processing events. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIG 3

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6 Mitochondrial Protein Homeostasis

mitochondrial proteins. Both Icp55 and Oct1 were shown tofacilitate cleavage of additional amino acid residues thatresults in exposure of a stabilizing amino acid residue at theN-terminus of the mitochondrial protein. This ultimately slowsdown the turn-over rate of the protein and demonstrates thatthe N-end rule is a universal cellular phenomenon presentalso in mitochondria (80,81).

Another important processing event takes place in theIMS. A few IMS proteins contain a presequence followed by ahydrophobic sorting signal resembling a transmembrane do-main (Fig. 3). The hydrophobic sorting signal facilitates proteinintegration into the inner mitochondrial membrane and ifremoved results in the release of a soluble IMS protein. Thebest characterized example of such a precursor is cytochromeb2 (30,32,83). The main player in this cleavage event is theIMS processing peptidase (IMP) (78,79). A few other substrateexamples include Ccp1 and Mgm1, which are cleaved by therhomboid protease, Pcp1. Mgm1, a conserved GTPase respon-sible for the fusion of the inner mitochondrial membrane isprocessed by Pcp1 to generate the IMS-soluble form. In highereukaryotes, the human ortholog of Mgm1, Opa1 undergoesanalogous processing. Several proteases have been reported toinfluence this event with the strongest evidence pointing onthe i-AAA protease from the AAAþ family and the metallopep-tidase Oma1 (84). Interestingly, independently of which prote-ase cleaves Opa1, the cleavage event leads to generation of ashort IMS form, which co-exists with the long membraneanchored form. The balance of short and long isoformsensures the maintenance of a morphologically healthy anddynamic mitochondrial population.

Processing events can also take place on the matrix side ofthe inner membrane. Of great importance is the processing ofone of the subunits of mitochondrial ribosome, MrpL32,executed by the m-AAA protease, also a member of the AAAþfamily (85,86). This processing event is necessary for the bio-genesis of functional mitochondrial ribosomes and defects inthis activity are directly linked to neuropathology. Thus, inaddition to enzymes, whose exclusive role is to perform matu-ration, mitochondrial proteases commonly associated withdegradation processes (discussed below), such as AAAþ pro-teins, take part in the proteolytic maturation of mitochondrialproteins.

Assembly of Mitochondrial Proteins

The biogenesis of mitochondrial proteins is completed by theirfolding and the assembly into multimeric complexes. A stringof classical chaperones and co-chaperones are present in themitochondrial matrix to assist in folding and to maintain pro-teins in a proper conformation (87,88). The representative ofall major families such as Hsp70 and its co-chaperones, Hsp60and Hsp100 (Hsp78 in mitochondria), are represented andthey display a broad specificity. However, more specializedchaperones such as those acting in the iron-sulfur cluster

assembly pathway are also known (89,90). It is less clear howproteins in the IMS are folded and assembled into the func-tional units. An important role is played by the MIA pathwaythat catalyzes the folding of IMS proteins by introducing disul-fide bonds. Recently, however Mia40 was discovered to act aschaperone that facilitates folding without utilizing cysteine oxi-dation chemistry (49,91). Furthermore, Yme1 that constitutesi-AAA protease was demonstrated to fold a protein targeted tothe IMS (92). Thus, the new functions assigned to the wellknown components of transport and turn-over pathwayspinpoint the direct interdependence between folding and otherbiogenesis events and quality control networks.

The inner membrane of mitochondria is exceptionally richin proteins. Within this membrane protein complexes repre-sent minimal functional units, however in many instancesinteractions between complexes are observed, which lead tothe formation of supramolecular assemblies. This tendency isbest exemplified by the packing of respiratory chain complexesand F1Fo-ATP synthase complex into supramolecular struc-tures, supercomplexes and oligomers (93,94). This organiza-tion may not only serve to achieve tight packing, but also inthe case of F1Fo-ATP synthase it leads to bending of the innermembrane and cristae formation, thereby increasing the sur-face area of inner membrane (94–97). Many proteins com-plexes are not evenly distributed in the inner mitochondrialmembrane and rather enriched either in the boundary innermembrane underneath the outer membrane or in the cristaeprotruding to the matrix (98–100). Interestingly, the recentlyidentified MINOS/MitOS/MICOS involved in maintaining thecristae architecture represents a key architectural componentof mitochondria (56–59,101), which may be involved indynamic processes that govern the distribution of protein com-plexes and are responsible for supramolecular organization ofmitochondria.

Degradation of Mitochondrial Proteins

Misfolded, non-assembled, and destroyed non-functional pro-teins pose a serious threat to cellular homeostasis. Mitochon-drial proteins are no exception to this rule. Thus far, we havedescribed the processes that govern mitochondrial proteintransport, sorting, maturation, and assembly. Glitches in anyone of these steps can occur and therefore the elaborate qual-ity control mechanism must exist to clear improperly targeted,unassembled, or damaged proteins (Fig. 4) (79,102,103). Mito-chondria have in place multiple quality control mechanisms.As a first line of defense a highly conserved, intraorganellarproteolytic system surveys protein quality control within theorganelle and aids in the removal of non-assembled andmisfolded proteins (Fig. 4). ATP-dependent oligomeric pro-teases mediate the complete degradation of dispensable mito-chondrial proteins and are found in the mitochondrial matrix(PIM1/Lon and ClpXP proteases), the inner membrane facingthe matrix (m-AAA protease), and the inner membrane facing

Varabyova et al. 7

the IMS (i-AAA protease). Both the i-AAA and m-AAA pro-teases extract proteins from the membranes for degradationbut also cleave both soluble IMS and matrix substrates,respectively. For instance, when IMS proteins such as thesmall Tim proteins (involved in protein transport) or Ups pro-teins (involved in lipid transfer) fail to properly fold or assem-ble, their removal is mediated by the i-AAA protease (70,104).These findings demonstrate the necessity to clear immatureproteins in the crowded mitochondrial compartments.

Additional peptidases, such as the inner membrane metal-lopeptidase Oma1 and the IMS localized Prd1 and Mop112extend the capacity of the mitochondrial proteolytic system(79,102). In addition to the quality control system localized inmitochondria, the role for the ubiquitin-proteasome system(UPS) in the turn-over of mitochondrial proteins is emerging(Fig. 4) (79,105). Outer mitochondrial membrane proteins,whose domains face the cytosol, are subjected to proteasomaldegradation, and this process is promoted by the recruitmentof the Cdc48 complex, a component involved in the ER-associ-ated degradation, upon stress (106–108). Furthermore, a set ofubiquitin ligases have been found on the mitochondrial surface(109,110). Remarkably, sporadic evidence suggests that theUPS is involved in clearance of mitochondrial precursor pro-teins during their prolonged stay in the cytosol (79,105).

The maturation of mitochondrial proteins by peptidasesgenerates a large number of peptides in the mitochondrialmatrix. One of the peptidases directly involved in the peptidedegradations is Cym1/PreP (111–113). The peptides generatedthrough proteolysis can also be exported outside mitochondriawith the help of peptide exporter of the inner mitochondrialmembrane. These peptides contribute to turning on cellularresponses to mitochondrial stress (114–116).

Another level of quality control is mitophagy, a processwhere damaged and non-functional mitochondria are removedvia a selective process involving the autophagosome(79,102,103). In this case, the entire content of mitochondriaincluding its proteins is subjected to lysosomal (vacuolar inyeast) degradation. The mitochondrial surface is residence tospecific receptors that are capable of targeting the autophagicmachinery to mitochondria (117–119). The E3 ubiquitin ligaseParkin and the PTEN-induced kinase 1 (PINK1) play a role inthe selective removal of depolarized mitochondria (109,120).Interestingly, mutations in the genes of both of these factorshave been linked to autosomal recessive forms of Parkinson’sdisease.

In conclusion, this review highlights the recent develop-ments, conceptual advances, and future challenges of this tre-mendously active research area. It is evident that protein

Protein degradation system in yeast mitochondria. Mitochondrial proteases are localized in the IMS, inner membrane (IM), and

matrix. Well known examples include; m-AAA, i-AAA, Pim1/Lon, and ClpXP from various mitochondrial compartments. Pro-

teins from the outer membrane (OM) are degraded by the ubiquitin-proteasome system in the cytosol. [Color figure can be

viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIG 4

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8 Mitochondrial Protein Homeostasis

import and late protein biogenesis events including proteolyticprocessing and assembly and protein quality control mecha-nisms are not distinct processes, but are intimately linked.Understanding these events both as individual processes, butalso within the context of an integrated cellular network, isvital since mitochondrial dysfunction is at the heart of neuro-degeneration and many other pathologies.

AcknowledgementsResearch in the AC laboratory is supported by the Foundationfor Polish Science—Welcome Programme co-financed by theEU within the European Regional Development Fund, EMBOInstallation grant, and the grants from Ministry of Science andHigher Education and National Science Centre in Poland. TheAV work was supported by the grant N N301 298337 fromMinistry of Science and Higher Education, Poland. Work in DSlaboratory is supported by the Australian Research Counciland the National Health and Medical Research Council.

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