mitochondrial import and the twin-pore translocase

REVIEWS

Mitochondria fulfil a large variety, and number, ofmetabolic tasks in eukaryotic cells. Among these are thegeneration of ATP through oxidative phosphorylation,the Krebs cycle, β-oxidation, the urea cycle, ketone-body synthesis, haem biosynthesis and amino-acidmetabolism. Besides these metabolic duties, mitochon-dria are also key players in other important pathwayssuch as programmed cell death, calcium signalling, andthe generation and detoxification of reactive oxygenspecies. At the molecular level, all these processes haveto be carried out by a large number of different pro-teins, and just how many proteins actually make up theorganelle has been a topic of speculation for a longtime. During the last year, detailed proteomic analyseson both the budding yeast Saccharomyces cerevisiae andhuman-heart mitochondria have been carried out,and they indicate that, respectively, these mitochon-dria contain about 800 and 1,500 different proteins thatparticipate in diverse processes1–4 (BOX 1). Nuclear DNAencodes most of these proteins (about 99%), whereasonly a minor fraction (8 proteins in budding yeast and13 in humans) is encoded by mitochondrial DNA.Accordingly, the cell needs to deliver these mitochondrialproteins to the organelle to satisfy the needs of itspowerhouse.

Similar to proteins that are destined for otherorganelles, such as chloroplasts or peroxisomes,mitochondrial proteins are synthesized on cytosolic

ribosomes and are subsequently transported to mito-chondria post-translationally5–12. Pore-forming multi-protein complexes — so-called ‘translocases’ — that arefound in the outer and inner mitochondrial membranethen take over and mediate further protein-transportsteps within the mitochondrion5,9–12. This article firstprovides an overview of the mitochondrial translocasesand then focuses on recent insights into the transport andmembrane insertion of a key class of hydrophobicmitochondrial proteins: multispanning, inner-mem-brane proteins. The mitochondrial inner membrane isparticularly rich in this kind of membrane protein,most of which are involved in various types of vitaltransport reaction. Among them are the metabolite car-riers and certain proteins that are constituents of theinner-membrane protein translocases.

The post-translational transport of such highlyhydrophobic proteins from the cytosol to the innermitochondrial membrane seems to be a challenge forthe cell, and we are only just beginning to understandthe underlying mechanisms. During their transport,hydrophobic segments need to be protected from aque-ous environments such as the cytosol and the inter-membrane space. They need to be moved across theouter mitochondrial membrane and through the inter-membrane space to reach the translocases in the innermembrane. Finally, at the level of the inner membrane,a driving force has to be provided by the organelle itself

MITOCHONDRIAL IMPORT ANDTHE TWIN-PORE TRANSLOCASEPeter Rehling, Katrin Brandner and Nikolaus Pfanner

The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most ofwhich mediate the vital transport of molecules between the matrix and the intermembranespace. The correct transport and membrane insertion of such proteins is essential formaintaining the correct exchange of molecules between mitochondria and the rest of the cell.Mitochondria contain several specific complexes — known as translocases — that translocateprecursor proteins. Recent analysis of the inner-membrane, twin-pore protein translocase (TIM22complex) allows a glimpse of the molecular mechanisms by which this machinery triggers proteininsertion using the membrane potential as an external driving force.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | JULY 2004 | 519

Institut für Biochemie undMolekularbiologie,Universität Freiburg,Hermann-Herder-Straße 7,D-79104 Freiburg,Germany.Correspondence to P.R.e-mail:[email protected]:10.1038/nrm1426

MEMBRANE POTENTIAL

The mitochondrial respiratorychain generates a protongradient across the innermitochondrial membrane thatleads to the formation of anelectrochemical potential. Thispotential is comprised of twocomponents — a chemicalpotential (∆pH) and anelectrical membrane potential(∆ψ). ∆ψ is the part of theelectrochemical potential thatfunctions as a driving force inprotein translocation.

520 | JULY 2004 | VOLUME 5 www.nature.com/reviews/molcellbio

R E V I E W S

membrane. The 450-kDa TOM complex thereforecontains receptor proteins that bind the targeting signals and direct the precursor proteins towards apore-forming core unit, which is known as the gen-eral-import pore (GIP)15–17. Electron-microscopyanalyses showed that the GIP complex could be foundin two different forms: one that has three pores and asecond one with only two pores15–17. An analysis of theGIP complex lacking the Tom22 or Tom20 receptorindicated that both proteins participate in the organi-zation of pores in the GIP complex17. Tom40, one ofthe seven constituents of the TOM complex, forms theactual protein-conducting pore in the outer mem-brane18 (FIG. 1). The TOM complex is involved in thetransport and insertion of outer-membrane proteins(BOX 2) and mediates the outer-membrane transloca-tion of inner-membrane proteins and matrix proteins(FIG. 1). In addition, Tom40 has recently been proposedto interact with incoming unfolded proteins in achaperone-like manner19.

Proteins that are destined for the matrix and someinner-membrane proteins are synthesized as precursorproteins that have an amino-terminal targeting signal, aso-called ‘presequence’. The positively charged prese-quence provides sufficient targeting information todirect a protein across the outer and inner mem-branes20,21. Accordingly, soluble proteins that function inthe mitochondrial matrix typically have a presequence(FIG. 1). Besides the matrix proteins, a group of inner-membrane proteins also uses presequences as a meansof targeting themselves to the inner-membrane translo-cation machinery. However, they usually possess furthersignals that direct them into the membrane9–11. Once thepresequences of both soluble and membrane proteinshave reached the matrix side of the inner membranethey are usually cleaved off by a specific protease thatresides in the mitochondrial matrix9–11. A second groupof inner-membrane proteins is transported in a differ-ent way. This group consists of multispanning proteinsof the inner membrane, which lack a presequence andinstead have several internal targeting signals12,22 (FIG. 1).Even though a consensus for the internal signalsequences has not yet been defined, it seems thatcharged and uncharged, short amino-acid stretches areused23. In addition, several of these signals in the proteinthat is to be imported function together to bind Tomreceptors23–25.

The inner-membrane presequence translocaseOnce a presequence emerges from the Tom40 pore, itengages the carboxy-terminal, intermembrane-spacedomain of the receptor Tom22 (REFS 26,27). From hereon, a translocase of the inner mitochondrial membrane(TIM) takes over and mediates the subsequent trans-port steps (FIG. 1). The presequence translocase is dedi-cated to the transport of mitochondrial proteins that usepresequences as a means of targeting. This multiproteincomplex is organized into two functional modules: amembrane-integrated translocase unit (the TIM23complex) and the presequence-translocase-associatedimport-motor complex (PAM complex).

to transfer the hydrophilic segments of the proteinacross the membrane and to insert the hydrophobic seg-ments into the lipid bilayer. Detailed biochemical analysesof protein transport and insertion are now providing uswith structural and mechanistic insights into theseprocesses. Here, we highlight these recent advances anddescribe the prevailing ideas that explain how mitochon-drial driving forces are used to insert multispanningmembrane proteins into the inner membrane.

Signals and outer-membrane transport Mitochondria are subdivided into four structural andfunctional units: two membranes (the outer and innermitochondrial membranes) and two aqueous phases— the intermembrane space, which separates themembranes, and the innermost compartment, whichis the mitochondrial matrix. All of these functionalunits need to receive the appropriate proteins thathave been synthesized in the cytosol. Signals in theprimary structure of the protein direct them to mito-chondria and subsequently to their destination withinthe organelle.

The initial recognition of targeting signals at theouter surface of mitochondria occurs through com-ponents of the translocase of the outer mitochondrialmembrane (TOM complex; FIG. 1)13,14. In addition torecognizing mitochondrial proteins, the TOM com-plex mediates their transport across or into the outer

Box 1 | The proteome of mitochondria

The importance of mitochondrial functions for cells, and the involvement ofmitochondrial dysfunction in several human diseases, has drawn much attention to theanalysis of the proteins that constitute the mitochondrion — that is, to analysis ofthe mitochondrial proteome. Depending on the species, tissue and metabolic state,mitochondria had originally been predicted to house between 1,000–2,000 differentproteins3. However, initial experimental analyses using two-dimensional gelelectrophoresis and mass spectrometry identified only a fraction of the mitochondrialproteins. A proteomic analysis of human-heart mitochondria using one-dimensionalSDS-PAGE2 revealed 615 different mitochondrial and mitochondria-associatedproteins, and thereby covered about 45% of the predicted proteome3. The analysis ofhighly pure mitochondria from the budding yeast Saccharomyces cerevisiae usingseveral protein-separation techniques and tandem mass spectrometry led to theidentification of 750 distinct proteins, which corresponds to a coverage of ~90% of thepredicted budding yeast mitochondrial proteome1. Both analyses highlighted proteinsthat could be classified into different functional categories based on homology or thepresence of conserved motifs. Most interestingly, about a quarter of the identifiedproteins in either study are of unknown function. So, these analyses highlighted anumber of novel mitochondrial constituents, which will be important for the futureanalysis of mitochondrial functions.

However, it will be difficult to obtain a complete view of the mitochondrial proteome,because it is continually adapting to different cellular requirements and certain proteinsare expressed in a tissue-specific manner. To tackle this problem, Mootha et al.79 havecarried out an integrated analysis that combined a proteomic analyses of mitochondriafrom different tissues (mouse brain, heart, kidney and liver) with genome-wide,expression-data analysis. Their analysis found 236 annotated proteins and 163 proteinsthat had not yet been annotated. At the protein level, 40% of the annotated proteinswere found in all of the four tissues that were analysed. Moreover, the available mRNAexpression data for 168 of the 236 annotated proteins indicated that 57% of the geneswere expressed in all four tissues. So, about half of the proteins seem to be differentiallyexpressed or, at least, to vary dramatically in their abundance79.


R E V I E W S

The first module consists of the three integral mem-brane proteins — Tim23, Tim17 and Tim50 (REFS 28–32;

FIG. 1). Although the role of Tim17 remains elusive,Tim23 has been shown to fulfil at least two functions.First, the amino terminus of Tim23 functions as areceptor for presequences on the intermembrane-spaceside of the inner membrane33,34. Second, Tim23 forms apore in the inner membrane of ~13-Å diameter throughwhich unfolded preproteins can be translocated acrossthe membrane35. Interestingly, the extreme amino termi-nus of Tim23 has been reported to span the outer mem-brane and was proposed to participate in aligning theTOM and TIM23 complexes during protein import36.However, a detailed analysis showed that the aminoterminus of Tim23 is dispensable for protein import,so a physiological function for this two-membrane-spanning topology remains to be determined37. Thethird component, Tim50, has a large domain in theintermembrane space, which binds to Tim23 (REFS 30,31).Tim50 seems to associate with presequence-containingproteins when they enter the intermembrane space andto direct them to the Tim23 pore (FIG. 1).

The membrane-integrated TIM23-translocasemodule uses the MEMBRANE POTENTIAL (∆ψ) across theinner membrane for protein translocation. The mem-brane potential has two distinct functions: it regulatesthe Tim23 pore35 and functions as a driving force thatenables the charged presequence to be transportedthrough the pore, across the inner membrane38.Interestingly, a recent analysis shows that the mem-brane potential can provide sufficient force to unfoldsome precursor proteins for transport39. However,complete translocation of the precursor across theinner membrane requires a second driving force. Thisis provided by the ATP-powered PAM complex thatcooperates tightly with the TIM23-translocase mod-ule in protein translocation (FIG. 1). The multiproteinPAM complex consists of mitochondrial heat-shockprotein-70 (mtHsp70) and its essential cofactors (BOX 3).Regulated cycles of mtHsp70 binding to the precursorprotein and its subsequent release provide an inward-directed force that allows precursor movementtowards the matrix. Once the precursor protein hasentered the matrix, it is generally converted to itsmature form by proteolytic cleavage of the prese-quence. The protease that catalyses this conversion isthe mitochondrial processing peptidase that residesin the matrix40,41 (FIG. 1). Once processed, the matureprotein is now ready to fold into its native conforma-tion and fulfil its function.

The carrier transport pathwayOuter-membrane transport steps. The route that mostmultispanning membrane proteins take to the innermembrane differs significantly from that taken bypresequence proteins that are destined for the matrix.Although a subset of multispanning inner-membraneproteins uses presequences (see below), most of themuse internal targeting signals and a specialized transport route that is known as the carrier pathway(FIG. 1). The most prominent members of this group of

Figure 1 | Protein-import pathways for mitochondrial proteins. Precursor proteins (brown)with positively charged amino-terminal presequences, β-barrel outer-membrane proteins (darkgreen), and multispanning inner-membrane proteins (blue) with internal targeting signals arerecognized by specific receptors of the translocase of the outer mitochondrial membrane (TOM)— that is, by Tom20, Tom22 and/or Tom70. Up to three dimers of Tom70 are recruited perprecursor (each Tom70 structure shown here represents a dimer). The precursor proteins arethen translocated through the Tom40 pore (the small Tom proteins of the TOM complex — Tom5,Tom6 and Tom7 — are not shown). The TOM complex contains two or three pores. The β-barrelproteins then require the small Tim proteins (Tim9–Tim10) to guide them through theintermembrane space, and the sorting and assembly machinery (SAM complex) for insertion andassembly into the outer membrane. Outer-membrane proteins with single transmembrane spanscan be directly inserted into the outer membrane by the TOM complex. Presequence-containingpreproteins use the presequence translocase of the inner mitochondrial membrane (the TIM23complex) for transport across the inner membrane. Tim23 forms a pore in the inner membrane.Presequence-containing inner membrane proteins can either be directly inserted into the innermembrane by the presequence translocase or be translocated to the matrix side and exportedinto the inner membrane107. It has been reported that the extreme amino terminus of Tim23spans the outer membrane36 (not shown). The membrane potential (∆ψ) and the function of thepresequence-translocase-associated import-motor (PAM) complex are essential for thetranslocation of presequence-containing proteins into the matrix. Mitochondrial heat-shockprotein-70 (mtHsp70) is the central motor component. It cooperates with Tim44, Pam16 andPam18 at the inner membrane and requires the matrix protein Mge1 (mitochondrial GrpE-relatedprotein-1) for nucleotide exchange. In the matrix, the mitochondrial processing peptidase (MPP)cleaves off the presequence. Multispanning inner-membrane proteins with internal signals requirethe Tim9–Tim10 complex for transport across the outer membrane and the intermembranespace. The insertion of these proteins into the inner membrane is catalysed by the twin-porecarrier translocase of the inner mitochondrial membrane (the TIM22 complex), which uses themembrane potential as an external driving force. This translocase contains two pores.

β-barrel outer-membrane protein

β-barrelprotein

Cytosol

Outer membrane

Intermembrane space

TOM complex

SAM complex

TIM22 complex

PAM complex

Tom70

Tom40

Tom22

Tom20

Innermembrane

Matrix

Tim23

Tim17TIM23 complex

Pam18Pam16

Tim44

Mge1

mtHsp70

+

–

∆ψ

C

N+ + +

+ + +

Inner-membraneprotein withinternal signals

Cytosolicchaperone

Matrix preproteinwith presequence

Tim9–Tim10

Tim9–Tim10

Inner-membraneprotein

Matrix protein

MPP

ATP

Tim54

Tim12

Tim22

Tim18

Tim50

ATP


R E V I E W S

aggregation by cytosolic chaperones of the HSP90 andHSP70 classes44. These chaperones are thought to shieldthe extensive hydrophobic stretches of the precursorsagainst the hydrophilic environment (FIG. 1).Subsequent delivery to mitochondria requires recog-nition of the carrier precursors at the mitochondrialsurface, and the precursor–chaperone complex bindsto a specific cytosol-exposed receptor molecule,Tom70 (REFS 24,44,45). Tom70 recognizes the internaltargeting signals of the precursor, which bind to a coredomain of this receptor23,25. In addition to the signal-binding site, Tom70 also provides an adjacent bindingsite for the chaperones44, because efficient precursorbinding to the receptor requires a cooperative interac-tion of both the precursor and the chaperone withTom70 (REF. 44; FIG. 1). It is interesting to note that up tothree dimers of Tom70 are recruited per precursor24,so the carrier-recognition step at the outer surface ofmitochondria results in the formation of a multiproteinassembly that is exposed to the cytosol46 (FIG. 1; BOX 4).

Subsequent transport steps require dissociation ofthe precursor from the receptor and the bound chaper-ones, a process that requires ATP (BOX 4). It is still notclear which protein actually binds ATP, but it is temptingto speculate that ATP binding by Hsp70 might con-tribute to the precursor release. During or after itsrelease, the precursor is passed to the Tom40 pore. Topass through Tom40, the precursor adopts a hairpin-likeconformation, which leaves the termini exposed to thecytosol while the loop tip, after having traversed themembrane, is exposed to the intermembranespace24,47. To achieve further translocation across theouter membrane, further factors are required. At astage when the precursor is still in the Tom40 pore, anessential protein complex of the intermembranespace — the Tim9–Tim10 complex — binds toit24,48–52 (FIG. 1; BOX 4). This 70-kDa complex — whichhas been proposed to represent a hexamer that is com-posed of three Tim9 and three Tim10 subunits — func-tions in a chaperone-like manner and binds tohydrophobic regions of the precursor as they emerge onthe intermembrane-space side of the membrane53.The Tim9–Tim10 complex mediates the transport ofthe carrier precursor across the intermembrane space,from the outer membrane to the inner-membraneprotein-insertion machinery, so the complex exists inboth a soluble and a membrane-associated pool50,51.Tim9 and Tim10 are required for the transport of car-rier proteins, although a closely related complex that isformed by the non-essential Tim8 and Tim13 sub-units also exists in the intermembrane space54. Onesubstrate has been found that requires this second com-plex for its transport under special conditions and thissubstrate is Tim23 (REFS 55,56). In addition, both theTim9–Tim10 complex and the Tim8–Tim13 complexare involved in the biogenesis of β-barrel-containingouter-membrane proteins57,58 (BOX 2).

All of these so-called ‘small Tim’ proteins havehighly conserved cysteine residues that resemble aZINC-FINGER MOTIF. However, the question of whether thecysteines coordinate zinc ions or if they, instead, form

mitochondrial proteins are metabolite carriers, suchas the ADP/ATP carrier, the dicarboxylate carrier andthe phosphate carrier, as well as the Tim23, Tim17and Tim22 components of the inner-membranetranslocases. As mentioned above, these proteins useinternal signals for their targeting. Early research onthe transport route that is taken by these proteins pre-dicted successive transport steps, each of which involvedifferent energetic requirements and protein com-ponents42,43 (BOX 4). Within the past ~15 years, all ofthese steps have been confirmed and characterized,and the protein components that participate in themhave been identified.

After the synthesis of carrier proteins in thecytosol, these hydrophobic proteins are maintained inan unfolded conformation and protected against

HSP90 CHAPERONES

Homodimer-formingchaperones that are composed ofsubunits that have a molecularmass of ~90 kDa. Each subunithas an ATPase domain. Hsp90proteins recognize specificsubstrate proteins and stabilizeintermediate folding states of theprotein.

Box 2 | The SAM complex of the mitochondrial outer membrane

Although a number of components that mediate matrix-protein transport and inner-membrane protein sorting have been identified, little is known about how proteins aresorted into the outer mitochondrial membrane. However, recent work has narrowedthis gap in our knowledge. Studies of the transport of outer-membrane proteins thathave several β-strands, such as translocase of the outer mitochondrial membrane(Tom)40 or porin, have led to the identification of a novel translocation machinery in the outer membrane. This SAM complex (sorting and assembly machinery) isspecifically required for the insertion and assembly of β-barrel proteins into the outer-membrane80 (see figure). Mas37, a peripheral outer-membrane protein, was identifiedas the first component of the SAM complex80. Two further constituents have beenfound since — Sam50 (also known as Tob55/Omp85)81–83 and Sam35 (REF. 84). Sam50and Sam35 are both essential for cell viability, which emphasizes a crucial role for theSAM complex in mitochondrial biogenesis. Sam50 is conserved from bacteria toeukaryotes, which indicates that the mechanism of protein insertion has beenmaintained throughout evolution81–83,85. Analyses of the biogenesis pathway ofmitochondrial outer-membrane proteins indicates that they are first translocatedacross the outer membrane by the TOM complex and are then inserted from theintermembrane-space side with the aid of SAM. This resembles the insertion pathwayfor bacterial outer-membrane proteins (outer-membrane proteins of bacteria areinserted from the periplasmic side of the membrane)80–84,86. Interestingly, the ‘smallTim’ (translocase of the inner mitochondrial membrane) proteins have been found toparticipate in this process by assisting precursor transport through the intermembranespace towards the SAM complex57,58.

β-barrelouter-membrane protein

β-barrelproteins

Cytosol

Outer membrane

Intermembrane spaceTOM complex

SAM complex

Sam50

Small Tims(Tim9–Tim10; Tim8–Tim13)

Tom70

Tom40

Tom22

Tom20

Cytosolicchaperone

Sam35Mas37


R E V I E W S

The TIM22 complex is organized into a peripheraland a membrane-integrated unit (FIG. 1). The periph-eral unit, which consists of the small Tim12 proteintogether with the Tim9–Tim10 complex 48–51, associ-ates with the integral portion of the complex on theintermembrane-space side. Although Tim9 and Tim10can dissociate from the complex, Tim12 is tightlybound and therefore functions as a docking point forthe predominantly soluble Tim9–Tim10 complex50,51.The membrane-integrated unit consists of three pro-teins: Tim18, Tim22 and Tim54 (REFS 63–68; FIG. 1).Little is known about the molecular roles of Tim18and Tim54 in protein insertion, but Tim22 wasrecently shown to form a pore in the innermembrane67. In an electrophysiological analysis, puri-fied and reconstituted Tim22 showed characteristicsof a single pore that has distinct conductance states,which reflect the characteristic open states of thispore. According to the measurements, a pore diameterof 12–17 Å, depending on the open state, was esti-mated67. The activation of the Tim22 pore in vitrorequires a high membrane potential (>140 mV) andthe presence of an internal targeting signal of a typicalprecursor. So, Tim22 combines two functions: it rec-ognizes internal targeting signals and responds to themembrane potential with rapid gating in a signal-dependent manner.

The three pores of the mitochondrial membrane-translocation complexes — Tom40, Tim23 and Tim22— therefore have different characteristic features.Whereas the Tom40 pore has a diameter of about 22 Å(REF. 18), the pore formed by Tim23 has a diameter ofonly 13 Å (REF. 35). These data indicate that the Tim23pore can only accommodate a single α-helical seg-ment of a preprotein in transit, whereas the pore sizeof Tom40 and Tim22 would allow them to hold twoα-helices at any one time. This is in agreement withthe observation that carrier proteins seem to traversethe TOM and TIM22 complexes in a hairpin-loopconformation24,47,69. Interestingly, outer- and inner-membrane pores respond differently to a membranepotential — whereas Tim23 and Tim22 are activatedby a membrane potential, the Tom40 pore is not18,35,67.This observation agrees with the fact that, in contrastto outer-membrane protein translocation, the inner-membrane TIM23 and TIM22 complexes use themembrane potential as a driving force for proteintransport.

In vivo, Tim22 is just one component of a multi-subunit translocation machinery and the behaviour ofthe full complex would be expected to differ from thatof the pore subunit alone. This is indeed supported bythe recent isolation and analysis of the TIM22 com-plex 68. A single-particle, electron-microscopy analysisof the 300-kDa TIM22 complex highlighted two stain-filled pits that are reminiscent of two pores, each witha diameter of about 16 Å. In agreement with this,after reconstitution of the entire TIM22 complex,two coupled pores could be discerned electrophysio-logically 68. Although both pores of the complexshowed the same basic characteristics that were seen

disulphide bonds is a controversial issue of research at present47,53,59–61.

The inner-membrane, twin-pore translocase. A special-ized translocation machinery — known as the carriertranslocase or TIM22 complex — handles carrier pre-cursors at the inner membrane (FIG. 1). This multiproteincomplex is solely dedicated to inserting multispanning,inner-membrane proteins that contain internal targetingsignals into the inner membrane. Interestingly, the onlyexternal energy source that is known to be required bythe TIM22 complex for this insertion process is themembrane potential (REFS 42,43,62).

HSP70 CHAPERONES

Molecular chaperones of about70 kDa that are composed of asubstrate-binding domain andan ATPase domain. Hsp70molecules interact withhydrophobic segments inunfolded proteins in an ATP-dependent manner and assist inprotein folding throughconsecutive rounds of substratebinding and release.

Box 3 | The presequence-translocase-associated import motor — PAM

The transport of proteins across the inner membrane into the matrix requires two energysources — the membrane potential (∆ψ) of the inner membrane (please refer to the maintext for further details) and the activity of the presequence-translocase-associatedimport-motor (PAM) complex. The mitochondrial heat-shock protein-70 (mtHsp70) isthe central component of the PAM complex and it binds directly to an incoming,unfolded polypeptide chain87,88. Translocase of the inner mitochondrial membrane(Tim)44, an essential peripheral inner-membrane protein, functions as a membrane anchor for mtHsp70 (REFS 89–93; see figure). The function of mtHsp70 has tobe tightly regulated to allow it to cycle between an ADP-bound state and an ATP-boundstate, which correspond to states of high and low substrate affinity, respectively. Thisregulation requires the cooperation of mtHsp70 with cofactors. DNAJ-LIKE PROTEINS

stimulate ATP hydrolysis by Hsp70 proteins, whereas a further exchange factor is neededto induce ADP release. Mitochondrial Mge1 (mitochondrial GrpE-related protein-1)9,10,11

functions as the exchange factor for mtHsp70, whereas Tim44 was thought to fulfil afunction that is similar to that of DnaJ-like proteins. Recently, though, Tim44 was shownto be unable to stimulate the ATPase activity of mtHsp70 (REFS 94,95). How, then, is theregulated cycling of mtHsp70 accomplished?

The answer to this problem lies in the discovery of Pam18 (also known as Tim14)94–96,anew component of the PAM complex. Pam18, an integral inner-membrane protein, isassociated with the presequence translocase and exposes a DnaJ-like domain to the matrixside of the inner membrane that stimulates the ATPase activity of mtHsp70 (REFS 94,95; seefigure). The discovery of Pam18 as a further component of the PAM complex indicatesthat previous models of how the motor translocates polypeptides were premature, becausethey were based on only three motor components (Tim44, mtHsp70 and Mge1). This isfurther underlined by the discovery of Pam16 (also known as Tim16), another essentialcomponent of the PAM complex, which cooperates with Pam18 in mtHsp70 regulationand seems to recruit Pam18 to the translocation machinery97,98 (see figure).

Tim17

Tim50

Intermembrane space

Inner membrane

Matrix

Matrix preproteinwith presequence

Tim23

TIM23 complex

PAM complex

Pam18Pam16

Tim44

Mge1

mtHsp70

+

+

–

++

∆ψ


R E V I E W S

for the Tim22 protein alone67, clear differences withregard to pore activation were evident. The TIM22complex had a higher sensitivity for the internal target-ing signal, as full activation of the complex required amuch lower membrane potential (~75 mV) thanTim22 alone. Interestingly, at this membrane potential,the targeting signal affects the two pores of the complexdifferently. Application of the targeting signal to thereconstituted TIM22 complex led to the rapid closureof one pore while simultaneously activating the otherto perform rapid gating transitions68.

So, what can we learn from these observations? Tounderstand the role of pore activation in the protein-insertion process, it is essential to find a way to correlatethe in vitro data with in organello transport. Althoughseveral transport intermediates along the pathway ofcarrier import have been known for a long time, it wasnot possible to obtain an intermediate of the stage ofprotein insertion into the inner membrane untilrecently68 (BOX 4). Using IONOPHORES to gradually reducethe membrane potential across the inner membrane inisolated mitochondria, it became possible to arrest a car-rier precursor in the TIM22 complex — that is, toobtain a stage-IV intermediate (BOXES 4,5).

By combining these observations with publisheddata, we are now able to propose a hypothetical modelfor how the twin-pore translocase uses the membranepotential to insert multispanning membrane proteinsinto the inner membrane.

Precursor tethering to the TIM22 complex. The mem-brane potential across the inner mitochondrial mem-brane is estimated to be 150–180 mV under normalconditions67. In vitro, the role of the membrane poten-tial can be dissected by modulating its magnitudeusing protonophores. When the membrane potentialis fully dissipated, protein import across and into theinner membrane is inhibited62 (FIG. 2a). In agreementwith this observation is the electrophysiological analy-sis of Tim22, as well as of the TIM22 complex, that hasshown that the pores are inactive in the absence of amembrane potential (REFS 67,68; FIG. 2a). Under theseconditions, the in vitro import of carrier proteinsinto isolated mitochondria seems to lead to two pop-ulations of precursors. One set of precursors arearrested such that they span the TOM complex andare associated with the Tim9–Tim10 com-plex24,46,48,49,70. A second set, however, is in contactwith the TIM22 complex48,49,68. This association seemsto be mediated by the interaction of the carrier pre-cursor with the Tim9–Tim10 complex, which isbound to the TIM22 complex. In addition toTim9–Tim10, the precursor is also close to Tim12under these conditions (chemical crosslinks betweencarrier precursors and Tim12 have been obtained)48,49.So, in the absence of a membrane potential, a set ofprecursors arrests at the step of outer-membranetranslocation, and a second set that has passed thisstage associates with the inner-membrane transloca-tion machinery. Therefore, more recently, the stage-IIIintermediate (BOX 4) has been subdivided into stage IIIa

Box 4 | The stages of carrier import into mitochondria

Initial analyses, which started in the 1980s, showed that the hydrophobic carrierprecursors are post-translationally imported into mitochondria99 and that theirtransport into the inner membrane requires the membrane potential (∆ψ) as an externaldriving force62,99. Our subsequent understanding of the transport pathway came fromdetailed in vitro import studies, which allowed the transport pathway to be dissected intofive distinct, successive steps (stages I–V; see figure). Each step represents a clearlydiscernable transport intermediate along the import pathway42.

The first step in transport (stage I) represents the soluble, cytosolic, chaperone-bound form of the precursor42,99. Subsequently, at stage II, the precursor is recognizedby receptors on the cytosolic face of mitochondria100. The carrier precursor can bearrested at stage II in the absence of ATP42,43. For the subsequent transport of theprecursor across the outer membrane, carrier precursors, as well as presequence-containing proteins, use the general-import pore (GIP) complex. The TOM complex(translocase of the outer mitochondrial membrane) contains receptor proteins thatbind the targeting signals and direct the precursor proteins towards this pore-formingcore unit or GIP complex. However, carrier proteins can be arrested at this transportstep if the membrane potential at the inner membrane is dissipated. This stage,stage III, was interpreted to represent the carrier when it is deeply inserted into theouter-membrane translocase (it is inaccessible to externally added protease)42 and isalready exposed to the intermembrane space101. Later it was realized that the stage-IIIintermediate is actually a mixture of two populations — one set of precursors interactswith the ‘small Tims’ (translocases of the inner mitochondrial membrane) inassociation with the outer membrane, whereas a second set of precursors is alreadytethered to the TIM22 complex.

It became clear that the insertion of the precursor into the inner membrane occursin a membrane-potential-dependent manner, but a pore-forming TIM complex wasnot considered at this time. At the inner-membrane insertion step (stage IV), nostable transport intermediate could be generated until recently68, so this stage wasinitially only inferred from kinetic analyses42,43. However, using ionophores togradually reduce the membrane potential across the inner membrane in isolatedmitochondria, it became possible to arrest a carrier precursor in the TIM22 complex— that is, to obtain a stage-IV intermediate. The insertion process ends with stage V,which represents the fully membrane-inserted carrier that has assembled into afunctional dimer.

Tom70

Cytosol

Outer membrane

Intermembrane space

TOM complex

TIM22 complex

Tom40

Tom22Tom20

Inner membrane

Matrix

+

–

∆ψ

Stage I

Stage V

Stage III

Stage IV

Stage II

Cytosolicchaperone

Tim9–Tim10

Tim9–Tim10Tim54

Tim22

Tim18

Tim12

ATP

Internal signal


R E V I E W S

complex68 (FIG. 2b). This change might reflect the dissocia-tion of the precursor from the small Tim proteins and itstransfer to other components of the translocase. This is inagreement with the idea that the low membrane potentialprovides the driving force for the movement of the pre-cursor within the TIM22 complex. However, the electro-physiological analysis of the TIM22 complex showed thatfull activation does not occur at such a low membranepotential (REF. 68). Accordingly, transfer of the precursorfrom the tethered to the docked state can occur withoutfull activation of the translocase. So, how can this discrep-ancy be explained?

Studies of the transport pathway of presequenceproteins have shown that the membrane potential hasa direct effect on positively charged presequences38.Negative charges on the matrix side of the innermembrane exert an ELECTROPHORETIC FORCE on the posi-tively charged presequence and allow it to moveacross the membrane through the pore that isformed by Tim23 of the presequence translocase38.Carrier proteins and other multispanning, inner-membrane proteins that use the TIM22 complex fortheir insertion have several positive charges in their

and stage IIIb to reflect these two scenarios9,11,12.With regard to the insertion process, the initial bind-ing of the precursor to the TIM22 complex in theabsence of a membrane potential has been called‘tethering’. The tethered precursor seems to be pref-erentially held at the translocase by hydrophobicinteractions, because it is largely resistant to saltextraction68, as would be expected if the hydrophobicsegments of the precursor are associated with theTim9–Tim10 complex52,53 (FIG. 2a).

Precursor docking in the TIM22 complex. The tetheredprecursor can be further transported along the insertionpathway when the membrane potential is fully re-established68. However, if the membrane potential iskept low in vitro (that is, below 60 mV) a second step inthe insertion process is reached — the docked stage. Thisdocked stage resembles the predicted stage-IV intermedi-ate (BOX 4). Similar to the tethered precursor, the dockedform is tightly associated with the TIM22 complex68.Atthis point, though,the precursor resides in a different mole-cular environment and ionic, rather than hydrophobic,interactions contribute to its association with the TIM22

ZINC-FINGER MOTIF

A motif in proteins that containsconserved cysteine residues.The sulphydryl groups of thecysteines coordinate a Zn2+ ion.

DNAJ-LIKE PROTEINS

Proteins that show similarity to aportion of the Escherichia coliDnaJ protein (the so-called J-domain) and that activate theATPase activity of Hsp70chaperones. The tripeptide His-Pro-Asp (HPD motif) iscrucial for the ATPase-stimulating activity of the J-domain.

IONOPHORES

Molecules that bind ions andthat allow their passage across amembrane barrier bysurrounding the charges duringthe passage of the ion across thelipophilic phase. Ionophores areusually specific for a defined setof ions — for example,protonophores are ionophoresthat are specific for H+.

ELECTROPHORETIC FORCE

Movement of a protein acrossthe inner mitochondrialmembrane can be driven by theelectric field that is generated bythe membrane potential (∆ψ).Positive charges in the proteinmove towards the negativelycharged side of the membranedue to the driving force of thefield.

Figure 2 | A hypothetical model for membrane insertion by the twin-pore translocase. Multispanning, inner-membraneproteins that have internal targeting signals are inserted into the inner membrane by the translocase of the inner mitochondrialmembrane (TIM)22 complex. The steps of transport can be experimentally dissected in vitro by modulating the membrane potential(∆ψ). a | In the absence of a membrane potential, the TIM22 complex is inactivated (red traffic light). However, initial binding of theprecursor to the translocase from the intermembrane-space side through the small Tim proteins Tim9–Tim10 and Tim12 can occur.This is the tethering step, and it resembles a subset of the stage-III-arrested carrier precursors (BOX 4). b | At a low membranepotential (less than 60 mV), the pores of the translocation machinery are maintained in a partially-activated state (orange traffic light),and the low membrane potential is sufficient to promote the insertion of one precursor loop into the translocase (the membranepotential exerts an electrophoretic force on positive charges that are found in the matrix-exposed loops of the precursor). This is thedocking step, and it resembles stage IV of carrier import. c | A high membrane potential (∆ψ; for example, 120 mV) across themembrane, together with the recognition of an internal targeting signal in the precursor, fully activates the translocase (green trafficlight). One pore closes tightly around the initially inserted terminal hairpin loop of the precursor, while the second pore mediates theinsertion of the next set of transmembrane domains. The process leads to membrane insertion, which occurs by the lateral openingof the translocase (insertion step).

Innermembrane

Matrix

+

–

∆ψ

C

+

–

∆ψ

C

Tim9–Tim10 Tim54

Tim12

Tim22

Tim18

C

TIM22 complex ++++++++

+++

+

NNN

Internal signal

Intermembrane space

a Tethering b Docking c Insertion

60

0 120

60

0 120

60

0 120

mV mV mV


R E V I E W S

stage-V product of carrier import42,43,46,62,68,69 (BOXES 4,5).It seems logical to assume that to move the docked pre-cursor into the membrane, the TIM22 complex needs toallow charged segments to be transferred across themembrane and into the matrix. Moreover, the complexhas to provide a hydrophobic environment for the futuretransmembrane segments of the precursor when theyare in the pore. Finally, the two Tim22 pores of theTIM22 complex need to fuse and one pore has to openlaterally to allow the membrane integration of all themembrane segments of the precursor.

In addition to the presence of a sufficiently highmembrane potential, a targeting signal in the precur-sor is required to fully activate the TIM22complex67,68, and one signal that activates the pore hasbeen identified in a carrier protein23,25. Interestingly,this signal resides at the beginning of the fifth trans-membrane segment, so it contacts the TIM22 com-plex when module III (that is, the region comprisingthe fifth and sixth transmembrane segments) hasbeen inserted into the translocase pore (FIG. 2b,c). Sucha contact can be achieved when the docked stage isreached. At full membrane potential, this signal acti-vates the complex, which leads to the closure of onepore and the rapid gating of the other68. With respectto the insertion process, an attractive — but stillhypothetical — scenario is that this initiates a

loops, which are involved in protein insertion into theinner membrane62,71–75. These loops, which have to tra-verse the membrane and are eventually exposed to thematrix, might have a role similar to that of prese-quences62. We suggest that the low membrane potentialis sufficient to promote the partial translocation of thecarrier into the translocase through an electrophoreticeffect on these loops62,68 — a mechanism that is similarto the one used by presequence-containing proteins38.However, although all of the loops contain positivecharges73–75, and are therefore, in principle, eligible topromote the docking stage, detailed analyses haveshown that the three matrix loops of a carrier precursorare functionally distinct. The carboxy-terminal thirdof the ADP/ATP carrier (module III), which consists ofthe last two transmembrane segments and their con-necting loop, probably fulfils the requirements forinsertion into the membrane alone, whereas other partsof the carrier depend on the presence of this modulefor their insertion69,76,77.

Precursor insertion into the inner membrane. A mem-brane potential across the inner membrane — forexample, 120 mV — provides enough external energyfor the precursor to be fully integrated into the innermembrane, where it can ultimately assemble into afunctional protein complex (FIG. 2c) — that is, into the

Box 5 | Analysing protein insertion into mitochondrial membranes

Analysing protein insertion into the mitochondrialmembranes is difficult in vivo, as kinetic analyses andbiochemical-fractionation techniques need to becombined. For mitochondrial protein transport, an in vitro import system — which imports in-vitro-synthezised, radiolabelled proteins into isolatedmitochondria and uses blue-native polyacrylamide gel electrophoresis (BN-PAGE) to address theformation of protein complexes102–104 — has provenhelpful (see figure). The latter technique is especiallysuited to the analysis of membrane-protein complexes that have been solubilized from membranes.If a radiolabelled, imported protein assembles into a multiprotein complex or into an oligomeric form (for example, stage V of carrier import; see figure andBOX 4), it is possible to separate and visualize suchcomplexes on the gel and therefore to assess the final stage of transport46,104. Moreover, in many cases, this approachhas allowed the identification of intermediates in the transport and assembly process, as the precursors cooperate withother proteins during transport and can therefore be found in different complexes of the pathway at different kineticstages24,46,68,70,80,82–84,86 (BOX 4).

BN-PAGE has also been found to be a helpful tool to visualize unknown intermediates in protein-insertionpathways68,70,80,86.As the precursors used in the import studies are imported in vitro, a further level of analysis can beapplied, because the formation of transport intermediates and of the membrane-integrated assembled complexes can beanalysed in a time-dependent manner and under different salt, temperature or energetic conditions. For example,membrane potential (∆ψ) can be modulated: full membrane potential leads to the membrane insertion and assembly ofthe carrier to form a dimer (stage V); dissipation of the membrane potential arrests the precursor at stage III of carrierimport (but this precursor is released from its associated components during BN-PAGE); and a low membrane potentialallows the precursor to be arrested at stage IV, in which it is found bound to the translocase of the inner mitochondrialmembrane (TIM)22 complex (see figure)46,68. One important technical development that can be used to address thecomposition of complexes following BN-PAGE is the so-called antibody-shift assay. The binding of antibodies to acomponent of the protein complex increases the size of the complex, which can easily be seen on the gel70,80,105,106.

Mob

ility

on b

lue-

nativ

e ge

l

Stage IV(300-kDa translocase–carriercomplex)

Stage V(mature carrierdimer)

Stage III(released carriermonomer)

∆ψ


R E V I E W S

potential functions in two ways to insert multispan-ning membrane proteins that have internal targetingsignals into the membrane — first, it has a directtranslocating effect on the precursor and, second, itactivates the insertion activity of the pores68.

Multispanning proteins with presequencesNot all nuclear-encoded, multispanning proteins of theinner membrane are imported by the carrier pathway

sequential insertion process. Module III of the carrieris inserted into the translocase and is retained in onepore that is induced to bind it tightly. Subsequently,the second pore mediates the insertion of the firstand second modules by rapid gating. However, theorder in which these two modules insert into the poreis unknown at present. Finally, we predict that theentire precursor is laterally released into the mem-brane. Therefore, we suggest that the membrane

Figure 3 | The transport of multispanning, inner-membrane proteins that have a presequence. Presequence-containing,multispanning, inner-membrane proteins are transported across the outer membrane through the translocase of the outermitochondrial membrane (TOM) complex and require the presequence translocase (the translocase of the inner mitochondrialmembrane (TIM)23 complex) for translocation across the intermembrane space and inner membrane. Both translocases have tocooperate tightly to promote precursor translocation. a | The absence of a membrane potential (∆ψ) at the inner membrane, togetherwith the inactivation of the ATP-driven, inner-membrane PAM complex (presequence translocase-associated motor complex), arreststhe precursor in the TOM complex (red traffic light). The intermembrane-space domain of the Tom22 receptor stabilizes this transportintermediate of the precursor in the TOM complex. b | A full membrane potential activates the membrane integral module of the TIM23complex and allows initial precursor insertion into the Tim23 pore. However, this is insufficient to promote inner-membranetranslocation of the precursor (orange traffic light). If the PAM complex is inactive, the precursor is maintained in association with theTOM complex. c | Transport of the precursor across the outer membrane and the intermembrane space requires both driving forces ofthe inner membrane — that is, membrane potential and the force provided by the ATP-driven PAM complex. The multispanningproteins can then enter the matrix space and be exported into the inner membrane107. Mge1, mitochondrial GrpE-related protein-1;MPP, mitochondrial processing peptidase; mtHsp70, mitochondrial heat-shock protein-70.

Tim17

Tim50

60

0 120

60

0 120

60

0 120

Intermembrane space

Matrix

TOM complex

Tom40

Tom22Tom20

Tim23

TIM23 complex

Pam18Pam16

Tim44

Mge1

mtHsp70

MPP

+

–

∆ψ

+

–

∆ψ

ATPPAMcomplex

Cytosol

Outer membrane

Inner membrane

a b c

Off On

Import motor

Off On

Import motor

Off On

Import motor+++

++

+

+++

mVmVmV


R E V I E W S

encounter similar problems with respect to their trans-port and insertion, cells have obviously evolved inde-pendent and mechanistically different ways to mediatetheir transport.

Conclusions and perspectivesResearch in recent years on protein insertion into mito-chondrial membranes has focused on the identificationof components that contribute to these processes. So far,all the constituents of the complexes that are involvedhave not yet been defined and new components havebeen identified only recently. However, for a full mecha-nistic understanding of the complex processes of proteintransport, it is essential to know the full set of compo-nents. Although studies on the composition of thetranslocation complexes themselves have led to the iden-tification of novel Tom, Sam, Tim and Pam proteins, it isprobable that more peripheral factors and regulatory fac-tors have been overlooked. It is hoped that these proteinswill be found among the large number of unknownmitochondrial proteins that have been identified in pro-teomic studies (BOX 1).

In addition to knowing the full complement oftransport-machinery components, it will be essentialto address their roles in transport and insertion at themolecular level. Central questions are, which proteinsrecognize the targeting signals and in what order dothe recognition events occur? Eventually, precursorbinding by a translocase has to be communicated to thepores, as has been seen for the activation of the TIM22complex. Comparing Tim22 alone with the TIM22complex indicates that not all of these functions canbe attributed to the pore-forming subunit, as thecomplex and Tim22 alone have different characteris-tics. Further analyses along these lines will eventuallyrequire more structural data on the translocases ofthe inner membrane and their subunits. Although theisolation of the inner-membrane translocases is animportant first step towards this goal, it has so faronly allowed a low-resolution electron-microscopyanalysis of one translocase. However, even this initialstructural information has provided importantinsights into functional aspects of the insertionprocess. In addition, analysis of the TIM22 complexthat had been reconstituted into lipid membranesproved to be important for our understanding of theinsertion processes. It is clear that more studies ofreconstituted translocases will be important to gainfurther mechanistic insights.

Finally, these structural and biophysical analyses indi-cate that the issue of protein insertion into a biologicalmembrane has reached a level of complexity thatrequires concerted technical efforts by both biochemistsand biophysicists to explain the molecular mechanismsinvolved. Nevertheless, the technical progress that hasbeen made in the analysis of the twin-pore proteintranslocase of the inner membrane — the TIM22 com-plex — will help us to further understand the mechanis-tic and structural aspects of other translocases, such asthe presequence translocase (TIM23 complex) of theinner membrane.

(TIM22 complex).A second group of multispanning pro-teins use a cleavable presequence as a means of targetingand therefore require the presequence translocase (TIM23complex) for transport into the inner membrane (FIG. 1).And, in many respects, the transport of these presequence-containing,multispanning proteins seems to differ signifi-cantly from that of proteins with internal signals78.

Although both classes of precursor use the TOMcomplex for transport across the outer membrane, thetransport pathways diverge as soon as the precursorsenter the intermembrane space. Carrier proteins arearrested in the TOM complex on inactivation of theTim9–Tim10 complex70. By contrast, the small Timproteins are dispensable for the transport of prese-quence-containing, multispanning membrane pro-teins across the outer membrane and the intermem-brane space78. A second significant difference regardingthe outer-membrane translocation of the two classes ofprecursor is the role of the membrane potential. Allmitochondrial proteins require the membrane poten-tial for transport across the inner membrane. However,dissipation of the membrane potential arrests the dif-ferent types of precursor at different transport steps. Inthe absence of membrane potential, carrier precursorsaccumulate at the Tim9–Tim10 complex24,48,49,70.However, this block in their transport does not pro-mote the formation of a stable intermediate with theTOM complex46,70,78. Quite the opposite is true for pre-sequence-containing, multispanning proteins. Theseproteins are stably arrested in the TOM complex underconditions of low membrane potential. In additionto their strict requirement for membrane potential tofacilitate outer-membrane translocation, only the pre-sequence-containing, multispanning proteins needthe PAM complex (BOX 3) at this transport step78. Bothforces together — the membrane potential of theinner membrane and the force provided by the ATP-driven PAM complex — seem to be required toactively pull the precursor out of the TOM complex(FIG. 3). Therefore, the transport of presequence-con-taining, multispanning proteins across the outermembrane and the intermembrane space is tightlycoupled to the function of the presequence translo-case (TIM23 complex).

In summary, the transport of multispanning pro-teins with internal signals across the outer membraneand the intermembrane space is a multistep process.Carriers use the soluble Tim9–Tim10 chaperone com-plex to pass through the outer membrane and inter-membrane space, and require a membrane potential asthe sole external energy source for their insertion.Unlike carriers, multispanning proteins that have a pre-sequence require two driving forces for their transportacross the outer membrane and intermembrane space— the membrane potential of the inner-membraneand the force provided by the ATP-driven PAM com-plex (FIG. 3). So, their translocation across the outer andinner membrane depends on a tight cooperationbetween the TOM and TIM translocases. Even thoughboth classes of multispanning protein have comparablebasic biophysical features and are therefore expected to


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AcknowledgmentsWe are grateful to A. E. Frazier for critical comments on the manu-script. Work from the authors’ laboratory is supported by theDeutsche Forschungsgemeinschaft, the Sonderforschungsbereich388 Freiburg, Max Planck Research Award, Alexander vonHumboldt Foundation, Bundesministerium für Bildung undForschung and the Fonds der Chemischen Industrie.

Competing interests statementThe authors declare that they have no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Saccharomyces genome database:http://www.yeastgenome.org/mtHsp70 | Pam16 | Pam18 | Sam35 | Sam50 | Tim8 | Tim9 | Tim 10 | Tim12 | Tim13 | Tim17 | Tim18 | Tim22 | Tim23 | Tim50 |Tim54 | Tom20 | Tom22 | Tom40 | Tom70Access to this links box is available online.

mitochondrial import and the twin-pore translocase

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