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A Novel Intermembrane SpaceTargeting Signal Docks Cysteines onto Mia40 during Mitochondrial Oxidative Folding Author(s): Dionisia P.

Sideris, Nikos Petrakis, Nitsa Katrakili, Despina Mikropoulou, Angelo Gallo, Simone Ciofi-Baffoni, Lucia Banci, Ivano Bertini, Kostas Tokatlidis Source: The Journal of Cell Biology, Vol. 187, No. 7 (Dec. 28, 2009), pp. 1007-1022 Published by: The Rockefeller University Press Stable URL: . Accessed: 12/07/2011 12:02
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ICSII;: Article




?ntermembrane space-targeting signal during mitochondrial cysteines onto Mia40 folding

P. Sideris,1'2 Nikos Petrakis,1,3Nitsa Katrakili,1 Despina Mikropoulou,12 Angelo Gallo,50 Simone Ciofi-Baffoni,5'6 Lucia Band,5,6 Ivano Bertini,56 and Kostas Tokatlidis1,4
Heraklion, Crete, Greece

of Molecular Biology and Biotechnology, Foundation forResearch and Technology Hellas, 7001 3 Heraklion, Crete, Greece 'Institute of Crete, 71003 department of Biology,department of Chemistry,and department ofMaterials Science and Technology, University of Florence, 50019 Sesto Fiorentino,Florence, Italy 5Magnetic Resonance Center and 6Departmentof Chemistry,University


imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative fold

ing. In this study,we show that the specific Cys of the is substrate de substrate involved indocking with Mia40 pendent,

cleft ofMia40 via hydrophobic mentaryto thesubstrate

We rationalize the affinity. a as dual receptor and an oxidase in a two step-specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by now docking of the substrate Cys juxtaposed to pair with function of Mia40 Mia40 the active Cys. interactions of micromolar

residues on the side of the docking Cys and dispensable on the other side, and (d) fits charged residues comple

an amphipathic with crucialhydrophobic helix (c) forms

Mia40 substrates. The ITSisa 9-aa presentin signal (ITS)

internal peptide and that (a) is upstream or downstream

the process being guided



IMS-targeting of

thedockingCys, (b) is sufficient forcrossingtheouter

membrane for targeting nonmitochondrial proteins,

Cys-containing proteins of themitochondrial intermembrane space (IMS) are oxidatively folded by Mia40 through the formation of transientmixed disulfides (Chacinska et al., 2004; Nao? et al., 2004; Mesecke et al., 2005; Rissler et al., 2005; Terziyska et al., 2005, 2007; Gabriel et al., 2007; Hell, 2008; M?ller et al., 2008; Reddehase et al., 2009). Mia40 also
functions known as a receptor recognizing evidence these substrates a by an un mechanism. Current supports Cys. For site-specific small

cleft that sits adjacent to the active site CPC motif and was proposed to be the substrate-binding domain. Evidence for

this proposal relied on mutagenesis ofMia40 residues in this cleft found to be lethal in vivo and decreased substratebind ing in vitro (Banci et al., 2009). However, it is still unknown
segments pairing. target the substrate toMia40 to allow correct covalent


via their N-terminal end Cys, which pairs with theC-terminal end Cys in an outer disulfide bridge in the folded state (Milenkovic et al., 2007; Sideris and Tokatlidis, Mia40 oxidizes the innerdisulfide pair of 2007). In contrast, COX 17, surprisinglyand unlike the small Tims (Banci et al., 2009). The exact Cox 17Cys used fordocking is stillunknown. The recent Mia40
Correspondence Abbreviations

Tims dock to Mia40


of the substrate-docking


we describe the signal for targeting In this study, different classes of substrates toMia40 (IMS-targeting signal [ITS]). A similar signal to the ITS was reportedbyMilenkovic et al. mitochondria IMS (2009) only for the small Tims and termed a Our data suggest partial overlap be sorting signal (MISS). tween the ITS andMISS sequences and reveal additional roles for the ITS not reportedbefore. To avoid confusion,we referto this signal asMISS/ITS. The MISS/ITS sentially allMia40 is a peptide of nine residues found in es substratesand primes one Cys fordocking

solution structureuncovered a hydrophobic

to Kostas Tokatlidis: used in this paper: ?-Me, ?-mercaptoethanol; BN, blue native; IMS, cyb2, cytochrome b^', DHFR, dihydrofolate reduc?ase; GAL, galactose; intermembrane space; ITC, isothermal titrationcalorimetry; ITS, IMS-targeting signal; MISS, mitochondria IMS-sorting signal; NTA, nitrilotriaceticacid; OM, outer membrane.

? 2009 Sideris et al. This article is distributedunder the termsof an Attribution Noncommercial-Share Alike-No Mirror Sites licenseforthe first sixmonthsafter thepub lication date (see Aftersixmonths it isavailable undera Creative Commons License (Attribution-Noncommercial-Share Alike 3.0 Unported license, as describedat http://creativecommons.Org/licenses/by-nc-sa/3.0/).

The Rockefeller Press $30.00 University j, Cell Biol Vol. 187No. 7 1007-1022 200905134 jcb.

1 007

with theMia40 active site CPC motif (Banci et al., 2009; is necessary and suffi Terziyska et al., 2009). The MISS/ITS cient for IMS targeting toMia40. Deleting it results in com
plete protein formation loss of import, whereas it to the organelle. across fusing The it to a nonmitochondrial ITS contains enough in (OM) targets


et al.,




clear in BN-PAGE
mutants were

intermediate(Fig. 1D, first panel; also confirmedby BN-PAGE in Fig. SI). The severe defect of the C45S mutant was also (Fig. SI,

in forming

the Mia40

lanes 9-11), whereas all other

for translocation

the outer membrane

independentlyofMia40, thus experimentally uncoupling these two processes. One crucial featureof MISS/ITS is that itforms a helix with vital aromatic and hydrophobic residues to the
same side of the docking Cys. Hydrophobic noncovalent inter

actions of this side of the MISS/ITS

chains of theMia40-binding Mia40 (sliding step), thus committing the crucial Cys for sub sequent covalent disulfide bonding withMia40
two-step mechanism no sequence they are solves similarity positioned are This the conundrum recognized of how sub

helix with aliphatic side cleft orient the substrate onto (docking step).
by Mia40 so that spe

In multiple Cys mutants of COX 17, theMia40 inter mediate was essentially abolished only when the thirdCys was mutated (mutantsC36/45S, C26/45/55S, and C36/45/55S; Fig. 1 E). Furthermore,when all other cysteines except C45 were mutated (C26/36/55S), covalent binding toMia40 was

strates with and explains

unaffected, confirming the crucial and specific role of C45 in Mia40 (Fig. 1E). docking to Previously, Heaton et al. (2000) showed that for yeast Coxl7, the fourthCys of the twin CX9C motifs (i.e., C57, which is equivalent toC55 of hCOX17) was essential in vivo. we addressedwhether yCoxl7 differedfromhCOX17 Therefore,



cific cysteines in each substratebond with the Mia40

Cys. Finally, we measured by calorimetry,

active site

for the first time, the

affinity involved in the crucial noncovalent interactions that guide the substrateontoMia40 via MISS/ITS.

Cys (C57), which is in agreementwith itsessential role in vivo

(Heaton et al., 2000).

in theCys docking to Mia40. The results (for single and multi mutants of ple yCoxl7) are shown inFig. S2.We find that inter action of yCoxl7 with Mia40, in fact, depends on the fourth

The data on human and yeast Cox 17 show thatCox 17 docks toMia40 differently from the small Tims. This is a
conundrum. How can different manner substrates yet be recognized different by Mia40 cysteines in a strict site-specific in each case? involving

Cox Cys

1 "7 docks specificity

onto from

l?iia4Q the

with snmail

a Tims



binding to the small Tims strictlydepends on their N-terminal end Cys of the twin CX3C motif (Milenkovic et al., 2007; Sideris and Tokatlidis, 2007), but it is unkn own whether this is true for Tim-unrelated proteins like Cox 17, which contains twin CX9C motifs (Arnesano et al., 2005; Banci et al., 2008) How does docking of Cox 17 onto
To address this, we generated single Cys to Ser mutants

dfeletsion targeting

analysis signal



reveals F^??a^O

an pathway


irar **h?:


Mia40 work?


for each Cys of theCX9C motifs of human COX 17, the sub strate studied before in the interactionwith MIA40 (Banci et al., 2009). Wild-type COX 17was importedefficientlyin iso lated yeastmitochondria (Fig. 1 A, lanes 3-7), and theoxidized
and reduced species were resolved, with of the oxidized the reduced form form. increasing with a concomitant decrease

active site Cys tomake the crucial intermolecular di sulfide.To identifysuch a putative targetingsignal,we applied N- or C-terminal end deletion analysis on Tim 10 (Fig. 2 A),
35S labeled the Tim 10 variants, was monitored (Fig. 2 B). and

thought that theremust be a specific targeting signal in the substrate that guides its precise positioning toMia40. In thisway, a defined substrateCys could be juxtaposed to the

typemitochondria (the lysate input controls are in Fig. S3).

Mia40 sensitive interaction by the presence Increasingly of a ?-Me longer dele intermediate



into wild

Additionally, a yMia40-COX17
ate was observed, was

mixed disulfide intermedi

with anti-Mia40

tions of theN-terminal end showed a defect inmaking the Mia40 intermediate,which was completely abolished when
residues 30-39 were deleted. In contrast, all C-terminal end

antibodies (Fig. 1A, lanes 8-10), and was ?-mercaptoethanol

(?-Me) sensitive, as expected. This intermediate was re


deletions (lacking 21, 33, or 43 residues) were unaffected.

Detecting the substrate released after interaction with Mia40,

2007; Sideris and Tokatlidis, 2007). This was DTT sensitive andMia40 specific,as clearing detergent-solubilizedmitochon
dria with anti-Mia40 antiserum beads (but not preimmune removed the 140-kD serum) band spe and protein A-Sepharose

solved also under native conditions in blue native (BN)-PAGE (Fig. 1B, 140-kD band; similar to smallTims; Milenkovic et al,

we saw thatAN 14 and ?N30 were released in oxidized form. In contrast, AC33 and AC43 still bound toMia40, but they
were stalled in this interaction and could not be released.

cifically but not the unassembled Cox 17 importedmonomer (Fig. 1B, lanes 4 and 5).
These assays were

Presumably, this is because they lack the crucial third and fourthCys (C61 and C65) thatpartnerwith C44 and C41 to make the folded oxidized species thus released from Mia40
(a) but the N-terminal end segment its own. 30-39 is crucial for target

(Sideris and Tokatlidis, 2007). This analysis suggested that ing toMia40
is not

tant of the CX9C motifs (Fig. 1 D). Only themutant in the third Cys (C45S), which pairs with the second one (C36-C45;
Fig. 1 C), was


for each




and (b) the region 14-30 facilitates targeting

on The loss of import capacity


tantsof theCys involved in copper(I) binding (C23 and C241;

* VOLUME 1B7 NUMBER ~7 * 20Q9







of the AN39 construct could be explained in two ways: (1) either it cannot cross theOM, or (2) it can cross theOM but can no longer bind toMia40. To investigate the latter,



kD 6745 26 14

10% M ~*





30' M aMia40

PI interm

660- mmm-



20' 40' aMia40



+DTT ?

C26 C36 -HH-1-1?H CC CX9C ]MIA40

C45 C55 CX9C







12 kD "10% 2 5
D _C23/24S


,~ ?.


10 20 30' '10% 2



10 20 30




-_ _

10 20 30' '10% 2


10 20 30


'10% 2

10 20 30' min

45 26 14 * V* E _C36/45S _C26/55S

*?--*.-?*l???5 10 20 30"10%



2 k? '-iqo/o
67; .*;

10 20 30' '10% 2

/U _?.

10 20 30' r??% 2

10 20 30' '10% 2

10 20 30'min


I i

(A) 35S-labeled COX 17 import and ?mmunoprecipitation as indi Figure 1. Differential requirement of COX17 Cys residues for interaction with Mia40. A but using BN-PAGE. (C) Cys organization of COX17. cated, followed by SDS-PAGE. (D) As inA for single Cys mutants. (E)As inD formultiply (B)As in Mia40 mutated Cys. (A7D, and E) Arrowheads indicate the mixed intermediate (interm).

we tested in vitro binding of theTim 10 constructs (in radio active form) to pureMia40 (bead immobilized; Fig. 2 C). The
results are identical to the

The small To


is conserved



binding when the first 30 residues are missing (AN30) and

complete loss of binding when a further nine residues are





test whether

the ITS

is conserved

in other




missing (AN39). To verify that the observed defect forAN39 is relevant in vivo, we directed this construct to the IMS by fusion to the presequence of cytochrome b2 (cyb2; residues 1-85), which targetsprecursors to the IMS independently ofMia40. In this way, any putative defects of AN39 in crossing the OM are bypassed, and this domain now targeted to the IMS can interactwith Mia40 in its physiological environment. The fusion cyb2(l-85)AN39Timl0
into both wild-type-

could be efficiently

reported previously to be importdeficient (Fig. 3 C; Lionaki et al., 2008), which could be caused by a defective import OM or defective interaction withMia40. As in this throughthe constructtheputative Tim 12 MISS/ITS is intact(residues 29-39; Fig. 3 A), we expected it to still bind toMia40 ifdirected to
the IMS as intermediate a cyb2 fusion. In fact, a time-dependent Mia40 was arrow seen for such a construct 3 D, (Fig.

responding deletions inTim9 and -12 were made. In Tim9, deleting 23 residues from theN terminus affected the inter action with Mia40 upon import to an extent, but deleting the (AN34) completely abolished the inter putative MISS/ITS action (Fig. 3, A and B). For Tim 12, the deletion AN28 was

chondria (Fig. 2 D) but did not give a Mia40 intermediate. This strongly argues that theAN39 deletion affects the inter action withMia40 inorganello, which is in agreementwith the
in vitro data.


and Mia40-depleted

mediates Therefore, the segment 30-39 of Tim 10 directly as a to Mia40 and functions binding Mia40-specific ITS. By the MISS comparison, signal reported by Milenkovic et al. (2009) was defined by residues 35-43.

When theputative MISS/ITS segmentbetween residues 29 and 39 was deleted inAN39Timl2 fused to the cyb2(l-85) pre
sequence, is necessary the interaction with Mia40 was

heads), which was abolished in Mia40-depleted mitochondria a from strain;Banci et al., (GAL)-Mia40 (prepared galactose withMia40. thattheAN28 can indeed interact 2009), confirming

MISS/ITS (Fig. 3 E), proving thatthe

for Mia40 binding. The

of Tim 12 segment 29-39

two cyb2 fusion constructs





for mitochondrial




et a!.


CX3C C40C44

CX3C C61C65 -93 AIM14 -93 AN30 -93 AN39 AC21 AC33 AC43 AC43 10 30 min





B kD

T10 AN14 10 30 10 30
_. *~ _ ~ ?

AN30 AN39 10 30 10 30

AC21 10 30

AC33 10 30

67-*~ 26

-<M?a40 ?nterm


-< monomer




T10 AN30 AN39 2 10 2 10 2 10 min

D cyb2(1-85)AN39Tim10


kD ~ 5% H " ^M?a4??nterm -


' 10 20 1

Mia40f mitos

5 10 201 min

Figure 2. Deletion analysis of Tim 10 reveals an internal targeting signal for theMia40 pathway. (A) Tim 10 truncations. (B) Importand analysis of Tim 10 versions as in Fig. 1 A. (C) GST-Mia40 pull-downs of different Tim 10 versions (as in Sideris and Tokatlidis, 2007). (D) Importof cyb2(l-85)AN39Timl0 in (Mia40j,; Band et al., 2009) mitochondria (mitos), ?nterm, intermediate. wild-type (wt) and Mia40-depleted

toAN28Timl2 and AN39Timl2 allowed us to uncouple the im Mia40 interactionand to test whether port to the IMS from the
this interaction is essential in vivo

in yeast cells (Fig. 3 F). cyb2(l-85)AN28, which bears the and interactswith Mia40, could restore viability MISS/ITS tional one. In contrast, thecyb2(l-85)AN39 MISS/ lacking the ITS and not interacting with Mia40 could not restore growth despite proper targetingto the IMS. of MISS/ITS
served Collectively, for Tim9 these data suggest that the presence that substantially, arguing that the interaction with Mia40 is a func

by complementation


inwhich residues of the MISS/ITS peptide 32-39 of Tim 10 were exchanged toAla (Fig. 4 A) and tested for Mia40 inter action (Fig. 4 B). Replacing M32 and N34 in the double Ala mutant (2Ala) only partially decreased the interactionwith
Mia40, seven which residues was were completely exchanged and abolished for Ala. when Because four, six, or aa the 2

thatwere mutated in the 4Ala mutant additionally to the

2Ala mutant were F33 K35, we tested these as

upstream of the N-terminal end Cys

and -12. Furthermore,

is con

in vivo.

interaction with Mia40



is essential

amino acid substitutions (Fig. 4, C and D). Exchanging F33 to Ala completely abolished theMia40 intermediate, whereas K35A had only a minor effect. L36A was affected to a substantial degree compared with the wild-type but


mediate in the F33A/L36A

to the effect of F33A residues and consensus motif of


not as critical

as F33.


double mutant can be attributed

alone. Identical results were


loss of




the MISS/ITS identify the critical residues for the functionalityof the MISS/ITS, Ala-scanning mutagenesis was performed (Fig. 4),
1010 JCB VOLUME 187 NUMBER 7 2009


with Mia40

obtained when the MISS/ITS mutants were used in Mia40 in translocation binding experiments mitoplasts (bypassing across the OM), which thus reflects a specific interaction (Fig. 4 E). Collectively, the Ala mutagenesis

CX3C C1 C2
23 34 28 39

CX3C C3 C4

87 87 87 109 109 109

Tim9 AN23 AN34 Tim12 AN28 AN39


W?????????????? -WW^V^ 100 -

Tim9 AN23Tim9 AN34Tim9 10 20' 1 10 201 ""l ?? 201 min '2

100 67

Tim12 AN28Tim12 T12 AN28 M M M 10%M " ,?Q% M HB*aw,?.~."'



+?Me Mia40| mitos _wtmitos_


E cyb2(1-85)AN28Tim12

kD 1


10 201 H
-? ? ^ 67

10 20!min . kD
^ 100


10 20 min

45 26 26 14


-?Me 104 103


-?Me 10 cells





103 102


is shown by an arrowhead. The TimlO ITS is conserved among small Tims. (A) Tim9 and -12 truncations. (B) The intermediate with Mia40 Figure 3. loaded as a standard. M, total mitochondria reisolated after import and protease (C) As in B for Tim 12 variants. 10% of in vitro translation mix was treatment. (D) Import of cyb2(l-85)AN28 Timl2 fusion as in B. (C and D) Arrowheads mixed intermediate, mitos, mitochondria; indicate theMia40 strain as de of Timl2 fusion variants using a GAL-Timl2 wt, wild type. (E) As in B but for the AN39Timl2 fragment. (F) In vivo complementation scribed previously (Lionaki et al., 2008). SGal, GAL media; SC, glucose media; WT, wild type.

? identified F33 (at position 7 relative to the docking Cys) and L36 (at position ?4) as the two importantresidues in the MISS/ITS, with F33 being absolutely critical. Both of these
residues are conserved (there are either identical or chem

Structural interaction

basis with

of Mia40




ically similar residues in these positions) in the consensus MISS/ITS described in sequence X[Ar]XX[Hy][Hy]XXC this study (Fig. 4 F and Fig. S3).

is the targetingfunction of theMISS/ITS linked to the requirement for Cys-specific docking onto Mia40? Because
recognizes substrates in a Cys-dependent manner, pre

cise positioning of the substrate Cys must be guided by a folded

MISS/ITS in a conformation-dependent manner to control



for mitochondrial




et a!.



Mia40. When projected ina helical wheel optimal attachmentto forms an amphipathic a helix with 5 the MISS/ITS (Fig. A), thedocking Cys sittingadjacent to thecritical aromatic residue of position ?7 on the same hydrophobic face that ismade by
the functionally important ? the hydrophilic 3, whereas in the crystal structure residues at positions charged ?7, ?4, and that side contains residues

B kD

wt 4Ala 6Ala 7Ala 2Ala '2 10 20''2 10 20"2 10 20''2 10 20"2 10 20'min

are dispensable for function.The MISS/ITS

of the human and

is indeed helical
yeast Tim9-Timl0

67-<**?->45 26

complexes (Webb et al., 2006; Baker et al., 2009). Although the

structure of Tim9 and -10 as they interact with Mia40 in structural terms is unavail

able, thehelical conformationofMISS/ITS

residues could explain

and thenatureof its


domain cleft ofMIA40 with thehydrophobic substrate-binding

14 (Banci et al., 2009). To test this concept, we

its specific


Mia40 cleft Tim9 and -10 docking to the reticalmodeling of the et the HADDOCK al., 2003). Only program (Dom?nguez using
one cluster of structures was obtained for both TIM9-MIA40


a theo

wtMFNKLVNNC K35A MFNALVNNC N38/N39AM FNKLVAAC F33A MANKLVNNC L36A MFNKAVNNC F33/L36AM ANKAVNNC L36A F33/L36A K35A N38/N39A F33A '2 10 20' '2 10 20' '2 10 20' '2 10 20' '2 10 20' min
^ ^

and TIM10-MIA40
200 structures with the mean structure

calculations containing 195 and 182 over

a backbone root mean region square of difference the proteins to of for the structured

D kD

1.5? and 1.3?, respectively. The buried surface area obtained ? the is 2,090 60 ?2 for TIM9-MIA40 complex and 1,471 ? are indicative of a very the 36 ?2 for TIM10-MIA40, which the ITS and thebinding between good surface complementarity of 5 cloud cleft ofMia40 (Fig. B, pink dots). The orientation of theMISS/ITS amphipathic helix of the hydrophobic face Tim residues interactionsbetween the functionally important (Fig. 5 B, gray) and hydrophobic residues of Mia40 (cyan). of the substrate-bindingcleftofMia40 Additionally, the length accommodates precisely the 9-aa longMISS/ITS helix, at the end of which thedocking Cys of the substrate engages in the Mia40 Cys (Fig. 5 B, yellow spheres). disulfide bond with the The modeling presented in this study supports the recognition of the MISS/ITS as a folded helix byMia40.
To scrutinize this concept is underpinned by aromatic-aromatic and aliphatic-aliphatic

45 26



E kD
67 45 26 100

wt L36A N38/N39A F33A K35A '2 10 20' '2 10 20' '2 10 20' '2 10 20' '2 10 2(Jmin

scanningmutagenesis of thedocking Cys of Tim 10 (C40). The underlying idea is that if the crucial docking Cys lieswithin an






downstreamwould not substantiallyaffectbinding to Mia40. In contrast, if thedocking works in the context of a foldedMISS/ ITS helix, moving theCys away from thewild-type position would put it ina completely opposite side of thehelix, strongly



it by up

to four residues



with the facing Cys of Mia40. Thus, affecting its interaction variants of Tim 10with a unique Cys in each of four positions
upstream or downstream

LYSNLVERC ScTim9 MFNKLVNNC ScTimlO VFNNILSTC ScTim12 (-4) (-3) (-7) X[Ar]XX[Hy][Hy]XXC consensus
Docking identifies critical residues of the ITS. (A) The Figure 4. Ala mutagenesis ITSof Tim 10 with Ala substitutions (bold). (B) Importsof Tim 10 Ala mutants performed as in Fig. 1. (C) Targeted Ala substitutions (bold) in the ITS of Tim 10. (D) Importassays of the mutants of C. (E) As inD, but precursors

were produced, startingfrom a Tim 10 inwhich full helix turn

of the wild-type



to span


Mia40 30-40

indicate the (B, D, and E) Arrowheads presented to mitoplasts. mixed intermediate. (F) Alignment of the ITS sequence (residues in Tim 10) in S. cerevisiae Tim9, upstream of the N-terminal C40 -10, and -12. The consensus ITS sequence and the Cys docking toMia40 are shown. The critical residues for the functionality of ITS are bolded, in with their position relative to the docking Cys in parentheses. Colons indicate that substitutions are observed, dicate that conserved periods

semiconserved substitutions are observed, and the asterisk indicates that in the residues or nucleotides in the column are identical in all sequences the alignment, wt, wild type.

1O? 2





"7 2009



(-4) (-3)


ITS of Til Tim 10

67 45 26

?2 ??"1 r2 ???1 rl kD^ 100




H2 To1


ri To1"? To1





m?n r2 ??1 r2 To1 ???1

-* Mia40 interm

K43C , , C44



-* monomer

250-, c I ? 200

1 160^
? 100 ? E 50 L36C V37C N38C N39C C40 -4 -2 -1 -3 0 Y41C K42C K43C C44 +1 +4 +2 +3

Figure 5. Cys-scanning mutagenesis and conformational properties of the ITS. (A) Helical wheel representation of ITS inS. cerevisiae Tim9, -10, and -12. The hydrophobic (black) and hydrophilic (gray) faces of the helix are indicated as half circles. (B) Theoretical modeling of hTIM9 and hTIMlO docking to TIMS and MIA40 are shown as red and blue ribbons, respectively. Hydrophobic residues ofMIA40 the hMIA40 substrate-binding cleft using HADDOCK. interactingwith TIMS are in cyan. Conserved aromatic and hydrophobic residues in the firsthelix of the TIMS interacting with theMIA40 hydrophobic cleft are in gray. Cysteines are inyellow, and the docking cysteines of the TIMS and MIA40 are shown as yellow spheres. (C) Cys substitutions inTim 10 and import and Mia40 intermediate (interm) analysis. Error bars represent standard error of the mean. (D) Quantification of theMia40 intermediate after 10-min import (three independent experiments).

Mia40 inter 42, 43, and 44 were tested for the formationof the mediate after import (Fig. 5 C). All of themutants completely

only thewild-type docking Cys (C40) was shown tomaintain the interaction with Mia40, as C40 is necessary and sufficient (Milenkovic et al., 2007; Sideris and Tokatlidis, 2007). The mu tants with theunique Cys inpositions 36, 37, 38, and 39 or 41,

all cysteines



to Ser. Previously,

a mutant


with Mia40, lost the ability to form a disulphide intermediate with the exception of C39, which showed an increased amount of the intermediate. Therefore, changes in thepositioning of the with docking C40 are generally not toleratedfor the interaction
Mia40. Overall,

docking depends on the folding ofMISS/ITS into a helix that Mia40. Cys of appropriately positions itopposite thepartner
signal fon mitochondria! oxidative folding Sideris et al. 1013

the Cys-scanning



that Cys


S.cerevisiae H.sapiens M.musculus B.taurus D.melanogaster C.elegans


COX17 (+3) (+4) (+7) CXX[Hy][Hy]XX[Ar]Xconsensus


wt COX17


m Mia40 interm

fromdifferent eukaryotes. Strictly conserved residues Figure 6. An ITS is present in the C-terminal end of Cox 17. (A) Alignment of Coxl 7 ITS sequences in the ITS consensus are shaded gray and bolded. Colons indicate that conserved substitutions are observed, and asterisks indicate that the residues or as in Fig. 5 A. (C) COX17 in the alignment. (B) Helical wheel projection for the ITS of COX17 nucleotides in the column are identical in all sequences mutants were imported and analyzed with Mia40 formixed disulfide intermediate (interm) (arrowhead), wt, wild type.


is present




of Cox i 7 Is thereaMISS/ITS present inCox 17 as well? Sequence align ment ofCox 17 and small Tims does not reveal aMISS/ITS up stream of theCOX 17 docking C45, as would be expected by end
analogy conserved to the Tims. However, such a motif became apparent distances

withMIA40 COX 17: (1) the reduced COX 17 shown to interact

by nuclear

a secondary structure content similar to the folded COX 17 (2) a theoretical docking between COX 17 andMIA40 similar to thatperformed in this study for the small Tims and Mia40 MISS/ supports a good surface complementarity between the ITS segment of COX17 andMIA40 (Banci et al., 2009). How can the MISS/ITS work in the case of yeast Cox 17
by circular dichroism (Banci et al., 2008), and




et al.,



as measured

downstream of theCOX 17 docking C45

residues in this segment were

(Fig. 6 A). Strictly

at equal

from the docking C45 as in the MISS/ITS of small Tims but downstream (positions 3, 4, and 7) and were thus aligned in the same face of the helical wheel projection with the dock ingCys (Fig. 6 B). Therefore, this importantstructuralfeature of theMISS/ITS is maintained. To testwhether theCOX 17 MISS/ITS is indeed functional, the Leu and He at positions 3 and 4 inCOX 17 (and conserved among Cox 17 sequences)
mutagenized. This double mutant could not form were a

MISS/ITS inwhich the docking Cys is the fourthone? In the and -4 the between for sequence yeast protein (Fig. 6 A, Cys3 first line), there is a Phe, He, and Tyr at positions 3, 4, and 7


intermediate (Fig. 6 C). At position 7, there is a Tyr in

cerevisiae Cox 17 (also maintained as a Phe/Tyr


Mia40, arguing for the importance of a hydrophobic/aromatic residue in position 7.We should note two things concerning
1014 JCB VOLUME 187 NUMBER "7 2009

in the small Tims at position ?7), but this is a His in all higher eukaryotic Cox 17 sequences. Mutating thisHis toAla in COX 17 in a tripleL48/I49/H52A mutant showed a strik with ing gain of function effectproducing more intermediate

of the small Tims (thathas no His inposition 7). the other residues in this region of the yeast Intriguingly, Cox 17 all form pairs of opposite charges (four pairs of Lys and Glu), which could interact through salt bridges and allow in the MISS/ITS thePhe, He, and Tyr to properly position the cleft ofMia40. In this case, the yCoxl7 MISS/ITS functions upstreamof thedocking C57. Therefore, itappears thatalthough there is aMISS/ITS inCox 17 that maintains thegeneral struc susMISS/ITS it functions downstream of tural features of theMISS/ITS, thedocking Cys for hCOX 17 or upstream of thedocking Cys foryCox 17.

the third Cys,


this region


to the consen

A 1


C40 C44 39 39 CX3C

C61 C65
.93 -?93

wtTimlO AN39Tim10 * AN39Tim10-ITS C40

OTTS end fusion toAN39Tim 10. wt, wild type. (B)AN39Tim 1 was for mixed disulfide inter imported and analyzed mediate (interm) formation with Mia40.

Figure 7. Swapping the ITS from theN- to the C-terminal end of Tim 10 does not affect targeting. (A) Schematic rep resentation of the Tim 10 ITS (zigzag line) and C-terminal

B kD 100 67

CX3C 1 2 5

93 10

10% 30

20 30 min
?. j*_ ^|^ja4Q ?nterm

i monomer

Docking calculations using the data-driven docking were performed to investigate thebasis program HADDOCK for selecting a differentCys in the Coxl7-Mia40 complex

IM- to does the not

C-terminal affect end targeting

from the
of TimlO

com performed on both the yeast and human Mia40-Coxl7 in which the the second of CPC motif of Mia40 plexes Cys was bound through a disulphide to either theC3 (Cys47 in COX17 or Cys45 in yCoxl7) or C4 (Cys57 inCOX17 or Cys55 in yCoxl7)

in yeast



to human.



The presence of the MISS/ITS

rather than at the N-terminal

at theC-terminal end of Cox 17

end, as for the small Tims, sug

of Cox 17, thus resulting in two types of com

organism. From the analysis of the data

gested that its targetingfunction is independentof localization within theprotein. Indeed, fusion of the ITS to theC terminus of the import-incompetentAN39TimlO (Fig. 7 A) restored MISS/ITS can exert its targetingfunction (Fig. 7 B). Thus, the of its localization within the targetedpolypep independently tide. However, despite efficient import and interactionwith Mia40, theAN39TimlO-ITS fusionwas rapidly degraded after import(in contrast towild-type TimlO), presumably because of incompleteor improperfolding.This behavior is reminiscentof the cyb2(l-85)AN39TimlO MISS/ (Fig. 2 D). Therefore, the ITS inAN39TimlO-ITS is sufficient for translocationacross the OM and fordocking to Mia40, but itcannot lead to productive can The latter MISS/ folding. apparently only occur when the ITS is properlypositioned N-terminally to theCX3C motifs.
MISS/ITS and to the can target Mia4Q import as well as its capacity to make a Mia40 intermediate



(Table SI), it emerges that the yMia40-yCoxl7 complex in volving C4 of yCoxl7 (HADDOCK score -152.7) has, by far,a much better score than thatinvolving C3 (cluster 1, HADDOCK
score -127.4; cluster 2, HADDOCK score -121.3). On the

C4 (cluster 1,HADDOCK
score ?56.0). Therefore, the experimental

C3 of hCoxl7 contrary,thehMia40-hCoxl7 complex involving score a score better than that involving has (HADDOCK ?88.7) score -66.6; cluster 2, HADDOCK
completely of the docking From data match the struc


of the best complexes for the two systems (i.e., yMia40?yCoxl7 complex involvingC4 of yCoxl7 vs. hMia40-hCoxl7 complex involving C3 of hCOX17), it ap that their different interactionmode is dictated by the pears nature and steric hindrance of the residues of the MISS/ITS stretch involved in the interactionwith the hydrophobic cleft of Mia40. Indeed, a Phe in yCoxl7 is changed to a Leu in hCOX17, and a Tyr inyCoxl7 is changed to a His inhCOX17. In particular, the Tyr in yCoxl7 is positioned close to two Phe residues of the Mia40 cleft, possibly engaging in strong
stacking interaction, thus

tural models

the comparison


independent proteins

nonmitochondrial organelle

for other substrates of theMia40

small Tims.

in yeast (Fig. S4). These results support the generality of the function of a MISS/ITS-dependent mechanism targeting


the C4



can target effi We wanted to know whether theMISS/ITS are not substrates for ciently (a) mitochondrial proteins that Mia40 and (b) nonmitochondrial proteins.We firstused Sue of ATPase (subunit e of yeast mitochondrial FIFoATPase), which is targeted to the innermembrane independently of Mia40 (Fig. 8 A; Tokatlidis et al., 1996; Everard-Gigot et al.,
2005). Deletion of the N-terminal end transmembrane anchor

pathway unrelated to the

MISS/ITS (AN23Sue) abolished importof Sue. Fusion of the of TimlO to theN-terminal end of AN23Sue (with Sue lack ing its unique endogenous C28 to avoid aberrant disulfides)
signal for mitochondnal oxidative folding Sideris et al. 1015


kD r'10% M


71 min 10 20


, r^ C

100 676745--? 26 14

10% M
., 4

ITS-AN23Sue "l 1 2 5 10 20 min

~?-pp* _ ... * -* ?._ *>? ... ? ^ m ia40 interm

M kD '10%
100 67 45 26

ITS-AN23Sue Mia4(H mitos

2 5 10 201min

4526 14-./


-?Me +?Me -?Me

.-?ITS-ANSue ^ANSue


^^^ J|fltJ!*$t fjWr*' +?Me



10% M

ITS-DHFR 5 10 20 min


F33A ITS-DHFR 10 20 min 5 10% M

F kD 100-

ITS-DHFRMia40t mitos ' 5 2 10 20 min '10% M ,i m




+?Me I


DHFR-ITS 10 20 min

InputPI aMia40

DHFR-ITS F33A 10 20 min kD 10% M 2 5

DHFR-ITS Mia40t mitos '""' " 2 10 20 'min 5 10% M





nonmitochondrial proteins. (A) Importof Sue in wild-type mito mitochondria. (D) ITS-DHFR importas inA. (E) As inD but with Mia40 indicates the fusion and import as inD. The arrowhead or preimmune serum (PI). (I)As in G but with the F33A Tim 10

substrates and Fusion of the ITS can target tomitochondrial Mia40-independent Figure 8. chondria. (B)As inA for ITS-DHFR. interm, intermediate. (C) As in B but in Mia40-depleted mitochondria. (G) DHFR-ITS the F33A Tim 10 ITSmutant. (F)As inD but in Mia40-depleted mixed intermediate. (H) Immunoprecipitation after the import of DHFR-ITS with anti-Mia40 As in G but in mitochondria. ITSmutant. (J) Mia40-depleted

restored import substantially (Fig. 8, A and B) and formed a ?-Me-sensitive Mia40 intermediate(Fig. 8 B) not observed for wild-type Sue (Fig. 8 A). Consistently, the ITS-AN23Sue im
port decreased

mitochondria when fusedwith ITS Fig. 8 D) was imported into to at itsN-terminal end, supporting the ability of MISS/ITS was not affected Mia40 could be observed (Fig. 8 D), import with mutant F33A ITS, which abolishes the interaction when the Mia40 (Fig. 4 D), was used (Fig. 8 E), and finally, import into Mia40-depleted mitochondria was unaffected (Fig. 8 F). This MISS/ITS ensured translocationacross suggested thatalthough withMia40 did not occur or was not stable the OM, interaction matrix. Indeed, this enough,which might lead to importinto the
was the case, as intramitochondrial localization of ITS-DHFR target DHFR across the OM. However, no intermediate with

in Mia40-depleted mitochondria (Fig. 8 C). The low butmea surable amount of ITS-AN23Sue still imported in this case Mia40 independent) is attributable to the ability (and is thus of the MISS/ITS to direct importacross theOM (see next two







withMia40 suggested localization paragraphs). As interaction in the IMS, we tested this directly (Fig. S5 A). The Mia40 intermediate and themonomeric ITS-AN23Sue were largely degraded inmitoplasts treatedwith protease, indicating that theyare accessible in the IMS. To address the capacity ofMISS/ITS to targetnonmito
chondrial folate proteins reduc?ase to the organelle, a small, (DHFR), we used mouse dihydro used well-folded As protein a control, fusion

Mia40 physiological substratefor

across the OM, which illustrates

mito (Fig. S5 C) showed it remained intact inprotease-treated to matrix the protein cpnlO). endogenous plasts (similar Thus, appending the ITS to theN-terminal end of a non
an important new function

for translocation is sufficient


of the nontargeting30 firstresidues of yeastADP/ATP carrier DHFR did not allow significant import(Fig. S5 B). N-terminallyto In contrast, a substantial amount of DHFR (around 10%;
101B JCB VOLUME 187 NUMBER 7 2009


as a model

in such experiments.

the MISS/ITS signal, which is apparentlyMia40 independent. OM translocation function is In authentic Mia40 substrates, the to the Mia40-dependent oxidative folding of the sub coupled
strate. However, for nonmitochondrial proteins, although the

ITS is sufficientforOM translocation, subsequent interaction with Mia40 is not always guaranteed. Conceivably, the ITS may not be accessible enough inwell-folded protein domains likeDHFR to bind toMia40. This can be rationalized on the basis that MISS/ITS is not present at the veryN-terminal end of physiological Mia40 substratesand thatsequences upstream of ITS could enhance the interaction withMia40. We alterna the ITS tively appended C-terminally toDHFR, as this could

major role of hydrophobic interactions in the binding process. These data provide thefirstexperimental support to theconcept occurs primarily through thatthe ITS-Mia40 interaction hydro
aromatic and aliphatic residues suggested by the theoretical

phobic interactions, underpinningthe packing of thecorresponding docking inFig. 5 B. They are also in full agreementwith previ ous mutagenesis of the Mia40 substrate-binding cleft (Banci

was Mia40 specific (Fig. 8 H), abolished when theF33A that mutant of ITS was used (Fig. 8 I), and absent from Mia40

allow better accessibility of the ITS, which is in linewith the result of AN39TimlO-ITS (Fig. 7). Indeed, theDHFR-ITS fusion protein interactedefficiently with Mia40 (Fig. 8, G-J). It formeda ?-Me-sensitive intermediate upon import(Fig. 8 G)

et al., 2009) and the mutagenesis of the substrateITS presented in this study.
The thermodynamic noncovalent interactions between

TimlO and Mia40

bond formation,

were analyzed by ITC. As ITC data can be dominated by the strong energetic contributionof covalent
thus masking the lower contribution of non

depleted mitochondria (Fig. 8 J). Intramitochondrial localiza tion experiments confirmed that DHFR-ITS is in fact localized in the IMS (Fig. S5 D). ?VSiSS/ITS?dependent binding to IVIia40

covalent interactions, AN290Mia40SPS was used as explained in the previous paragraph. TimlO was reduced as shown by AMS thiol-trapping experiments (Fig. S5 E). A clear inter action between AN290Mia40SPS (injected) andwild-typeTimlO (in the cuvette) was detected with a measured Kd of 3.3 uM (3.3 x 10-6 ? 0.7 x 10"6M; AG -7.42 kcal/mol; Fig. 9 D,


mediated micromo?ar


hydrophobic affinity



helix (supportedby the theoretical modeling and the a to all Cys mutagenesis experiments) point key role of hydro

The discovery of the ITS targetingto Mia40, the crucial role of the hydrophobic residues as shown by mutagenesis, and the structuralrequirement for these to be on the same side of the

which is consistentwith a specific interactionin vivo. top left), Deletion of the first30 residues of TimlO (AN30Timl0) low ered the affinity19-fold (Kd-63 uM; 6.29 x 10"5 ? 0.14 x 10-5M; AG -5.68 kcal/mol; Fig. 9 D, topright), a value still

consistent with a physiologically relevant interaction. However, deletion of a further nine residues, thus entirely cleaving the

hydrophobic interactionsmust be strong enough to properly orient the substrateso thatdisulfide bonding can thenoccur in a
the importance of these noncovalent hydrophobic interactions,

in this targeting




second step locking the interaction withMia40. To directly test

ITS, completely abolished the interaction(Fig. 9 D, bottom left; background binding with Mia40 injected alone to the buffer solutionwithout any substrate inFig. 9 D, bottom right).These results provide the firstdirectmeasurement of the affinity of
the noncovalent interaction between Mia40 and one of its sub

we (a) evaluated in binding experiments in vitro the effectof salt and detergentand (b)measured the thermodynamicsof the by iso thermal titration For these calorimetry (ITC). experiments,we used AN290Mia40SPS, which (a) lacks the first290 residues that are not importantfor interactionand dispensable in vivo ITS-mediated interactionof the substratewith Mia40

versions of TimlO withMia40

strates,are in agreementwith the binding data (Fig. 9 A), and correlatewell with the import and interactionof the relevant inorganello (Fig. 2 B).

Previous analyses of theMia40-dependent oxidative folding showed that Mia40 recognizes specific cysteines in this pro cess based on studieswith the small Tims showing thatthefirst

(unpublished data) and (b) has both cysteines of the active site CPC exchanged to Ser so that it maintains its capacity fornon
covalent interaction with the substrate without

sequent covalent disulfide bonding with the substrate. ontopure, activelylabeledprecursors His-tagged AN290Mia40SPS immobilized on Ni beads. The results (Fig. 9 A) are in agree ment with those from import assays. Wild-type TimlO binds substantiallyas expected; deletion of thefirst30 residuesweak ens binding to some extent,but entirelyeliminating MISS/ITS were ob abolishes Similar results (AN39) completely binding.
Binding in vitro was tested in pull-downs of different radio


the sub

Cys of theCX3C motifs is crucial for docking (Milenkovic et al., 2007; Sideris and Tokatlidis, 2007). Binding of other sub
strates remained a conundrum. We report the surprising finding

thatCoxl7, a CX9C substrate,docks to Mia40 via its thirdor fourth of the twin CX9C which is in sharp contrast motifs, Cys
to the small Tims. This clear difference can now be rational

fused toAN23Sue. Because this in vitro assay fornoncovalent Mia40 faithfully reflectsbinding inor binding of precursors to we 2 and tested theeffectof salt and detergent 8), ganello (Figs. in thisassay. Salt did not seem to influence the interaction up to 500 mM NaCl (Fig. 9 B), confirmingthat ionic interactionsare
crucial. In contrast, increasing detergent concentration

tainedwith theAN23Sue precursor, for which substantialbind was was N-terminally detected when the MISS/ITS ing only

Mia40. The ITS has several defined features. It is a 9-aa pep tide that is conserved among small Tims immediatelyupstream

ized in the lightof our identification of a novel targetingsignal that is conserved small Tims and Cox 17 and (the ITS) among allows a particularCys, differentin each substrate, to dock to

of their N-terminal end firstCys. The consensus sequence is X[Ar]XX[Hy] [Hy]XXC. The Ar residue at position -7 relative to the first Cys is crucial as singlemutagenesis of this residue abolishes targeting. The Hy at position ?4
crucial. The ITS tends to form but not a helix that aligns un

is also important


weakened the interactionsubstantially (Fig. 9 C), arguing for a

charged/hydrophobicresidues (in particularAr and Hy at -7 and -4 and -3) to the same side of the crucial docking Cys.
Targeting signal for mitochondria! oxidative folding * Sideris et al. 1017

r14- 10%



TimlO WT AN30 +1 10% *"13.6

AN39 +' 10%'1.3 0.2


AN23 Sue '-ITS 10% r+1

+ ITS 1 +1 Mia40 10% ^


% Binding




50 B 150 500 NaCI (mM) C kD +1 r+1 Mia40 10% ""^ +1 r14


0.01 +'

0.05 '- +' '^KP 4.1

0.1 '- +'

^VHr 8.6

0.5 Triton (%) '- +'Mia40 ' ^P^ ?&?t' 1.6 1.3

% Binding




% Binding

Time (min) 0 10 20 30 40 50 60 70 80
0.00 -0.10 -0.20 -0.30

Time (min) -10 0 10 20 30 40 50 60 70 80


AN30 TimlO


1.0 0.5 1.5 Molar Ratio



' ' ' o'.O o'.5 10 l'.5 ?0

Molar Ratio

Time (min) -10 0 10 20 30 40 50 60 70 80 0.00 i

^-0.05 CD ?-0.10 -0.15 - 0.20 -

Time (min) -10 0 10 20 30 40 50 60 70 80

i i i ' i i ' i ' i ? i i

1-1.5 I-1.7J O
E-1.9-1 75 o * -2.1 -



1.0 1.5 Molar Ratio




0.5 1.5 1.0 Molar Ratio



The noncovalent ITS-dependent binding of substrates to Mia40 is mediated by hydrophobic interactions. (A) Purified His-tagged AN290Mia40SPS Figure 9. was bound toNi2+-NTA beads. 35S-labeled proteins were incubated with Ni-NTA beads with or without AN290Mia40SPS at 15?C for 2 h. (B) 35S-labeled TimlO was AN290Mia40SPS incubated with Ni-NTA bead-immobilized for 2 h at 30?C in the presence of 50, 150, or 500 mM NaCI. (C) As in B, but alone (bottom right) or with binding was performed in the presence of 0.01, 0.05, 0.1, or 0.5% Triton X-100. (D) ITC of 0.25 mM AN290Mia40SPS

or Tim Tim Tim 10 (topleft), 10AN30 (topright), 10AN39 (bottom 0.025 mMofwild-type (WT) left).







theoretical docking of the ITS onto the substrate-binding cleft ofMia40 shows (a) very good surface complementarity (buried surface area of 1,471?2 or 2,090 ?2 forTIM9-Mia40

feature with count

the typical matrix-targeting of ITS to cross

signal, the OM.



ac the

and TIM10-Mia40 complexes, respectively) and (b) stabilizing hydrophobic interactionsbetween the crucial elements of ITS and aliphatic side chains in the substrate cleft ofMia40. Cys
scanning experiments

C45 ofCOX 17, identifying itjust downstreamofC45, which is a result. striking Underscoring the importanceof theprecise nature we found that inyCoxl7, this of the residues in the ITS stretch, a is oriented in such in thecleftofMia40 thatthe last way signal Cys of the twinCX9C motif is primed for docking toMia40. The identification of the ITS forCox 17 agrees with the theoreti cal docking fora putative complex between COX 17 andMIA40

with Mia40. This cept that the ITS must fold in its interaction ratherthan simply sequential, featureof the ITS led structural, us to look for such a signal in the vicinity of the docking Cys




to the con

ITS ismuch shorter,and its functionality reliesmainly on the hydrophobic residues and the crucial Cys in contrast to the matrix-targeting signal,where both hydrophobicity and charge are important (Abe et al., 2000). One other ITS was reported for the cytochrome c heme lyase but is not present in other proteins and has very differentproperties from the ITS (^60 residues long and stronglyhydrophilic; Diekert et al., 1999). During preparationof ourmanuscript,Milenkovic et al. (2009) reported the identification of theMISS targeting signal in Tim9 and -10. This partially overlaps with the ITS signal se with good agreement between quence reported in this study, the data. In our study, identificationof ITS in the small Tims provided the startingpoint to additionally (a) identifysuch a

for the capacity

In contrast,

sequence is more accessible for binding to Mia40. The N-terminal end fusion ITS-DHFR retained the ability for com plete translocationacross theOM, illustratingthisnew role for the ITS, which is Mia40 independent.This analysis allowed us
of translocation across the OM and to uncouple the two events

cient for targeting.The results were more compelling in the case of a C-terminal fusion of the ITS toDHFR, presumably because in this case (as in the AN23TIM10-ITS), the ITS

geting indeedproved thispoint. Further,fusing the ITS to either a mitochondrial protein that is not a substrate for Mia40 (Sue ofATPase) or a nonmitochondrial protein (DHFR) was suffi

of theITS (9 aa) fitsexactly to the substrate-binding cleft,ending up in thecrucialCys pair between the substrateandMia40. The variable positioning of the ITS in different Mia40 substrates suggested that itfunctions independently.Swapping the ITS to theC-terminal end of TimlO without affecting tar

both Tims and Coxl7 are ori (Banci et al., 2009). Interestingly, ented in the Mia40 substratecleft similarlyand perpendicularly to the twohelices a2 and a3 of the Mia40 core.Also, the length

signal in differentsubstrates, (b) solve the conundrum of how differentsubstrates are recognized equally well byMia40, and (c) dissect the structuraland biophysical basis of this recogni
tion process. The noncovalent protein-protein interactions that are cru

cial for Mia40 binding and precede the covalent Cys docking were found to be primarily hydrophobic, as theywere not

affected by salt but were substantially competed by detergent in vitro. These results are in linewith the theoreticaldocking strates (Tims or Cox 17) with Mia40,
of interacting residues seems for a putative complex of different types of sub


in which hydrophobic
to play a major role.


We measured for the firsttime the affinityof the non covalent binding of the substrate to Mia40 by ITC. Itwas found that TimlO had a strongnoncovalent thermodynamicaffinity to a of TimlO byMia40 Mia40, indicating specific recognition
before sense the redox chemistry as can take place. This also makes of a thermodynamically, the subsequent interaction

folded (oxidized) TimlO with itspartnerTim9 is two orders of with a Kd of 30 nM (Lu et al., 2004), and magnitude stronger
thus, the entire process can be

Mia40-dependent folding, which are tightlycoupled for au thentic Mia40 substrates. Having identifiedthe ITS as the crucial targetingelement in the Mia40 pathway,we checked for ITS-like signals in the known and predicted substratesofMia40 (Table S2). The crite ria fordefininga sequence as a putative ITS were (a) a lengthof 9 aa upstream or downstream of theCys and (b) thepresence of
one aromatic and one hydrophobic residue at positions 4 and 7.

yeast and human Cox 17, theputative ITS identifiedinTable S2 may be oriented in different ways in the cleft ofMia40. In any case, though, this signal primes a unique Cys for interaction with Mia40. Therefore, it appears that the presence of ITS is

For the 15 known/predicted Mia40 substrates (Gabriel et al., 2007), we discovered at least one ITS identified (Table S2), with the exception of only yTiml3. In lightof the results for

MISS/ITS structurefor optimal inter potentially stabilize the action within the Mia40 cleft. We propose a working model (Fig. 10) whereby the MISS/ITS has a dual function: first it guides the precursors through the OM, and then it orients them via hydrophobic interactionsto thecleftof Mia40. The latterreflectstherecogni tion byMia40

cascade of increasing affinities.The initial interaction that Mia40 ismainly caused by the MISS/ guides the substrate to ITS, as cleavage of this segment (AN39Timl0) completely abolishes binding. The fact thatbinding is decreased but still measurable forAN30 suggests that the upstream 30 residues




whereas inCX9C
ence for priming

quite general. Furthermore, itappears that inCX3C substrates, ITS signals prime preferentially the firstCys for docking,

butwith a prefer substrates,this is less strict

of the C-terminal end cysteines.

recognition process, which thereby allows the correct disul fide to be formed (step 2: docking step,Cys dependent and

(step 1: sliding step, ITS guided and noncova and lent) provides thebasis for the receptor functionofMia40 in the IMS. Juxtapositionof the correct substrate-dockingCys to the active siteCys ofMia40 is a consequence of this initial

The ITS expands the repertoire ofmitochondrial-targeting Its to an form signals. propensity amphipathichelix is a common

covalently bound). Correct Cys priming in each substrate as determined by the ITS provides a mechanistic framework for thekey role ofMia40 in the mitochondrial oxidative fold
ing pathway.



for mitochondnal



* Sideris

et al.





step 1 ITS dependent step 2
cysteine covalent dependent interaction release/folding

non-covalent IM


(step 1, sliding) The substrate slides onto Mia40, where it is oriented by Figure 10. Sliding-docking model for the interaction of substrates with Mia40. interactions in the cleft of Mia40. The correct Cys of the substrate is thus primed to make the theMISS/ITS through noncovalent, mainly hydrophobic via the covalent mixed disulfide bond between the substrate-docking Cys and disulfide with Mia40. (step 2, docking) The substrate now docks onto Mia40 a the juxtaposed active site Cys ofMia40. Finally, complete oxidation releases the substrate in folded state. IM, innermembrane.




Isolation of mitochondria iso mitochondria were and M/a40-depleted mitochondria Wild-type lated from the S. cerevisiae strain D273-10B (MAT-a) and from theGAL strain as described previously (Daum et al., 1982; Glick, 1991; Mia40 in medium Banci et al., 2009) The yeast strains were grown at 30?C and 1% (wt/vol) yeast extract, 2% (wt/vol) bacto-peptone, containing to 5.5 2% lactate (vol/vol) pH adjusted (YPL). In the case of the GAL to maintain the suppression of Mia40 strain, 0.2% glucose was added the endogenous gene.

AN290 translation purposes. Yeast M?a40 containing a double Cys to Ser substitution at the CPC catalytic center was cloned in pET22 (EMD) for bacterial periplasmic expression with a C-terminal His6 tag by PCR from pGEX/yMia40SPS (Band et al., 2009). Amino acid substitutions for and yeast TimlO were generated by PCR-based COX17, yeast Coxl7, site-directed mutagenesis kit;Agi site-directed mutagenesis (QuikChange from pSP64-based lent Technologies) plasmids containing the COX17, and yeast Tim 10 gene, respectively. Primer design and the yeast Coxl7, to the manufacturer's guide PCR conditions were performed according reactions. lines. The mutations were verified by sequencing

Generation of mutant substrate proteins Truncated versions of yeast Tim 10, -9, and -12 were amplified by PCR and cloned into a pSP64 vector (Promega) downstream of the SP6 promoter for in vitro transcription/translation purposes. For the N-terminal deletions, a Met was added at position 1. To improve 35S labeling, three methionines were added at the C terminus where necessary. The sequence encod ing the first85 aa of cyb2, which constitutes its IMS-targeting sequence (Glick et al., 1992; Beasley et al., 1993; Schwarz et al., 1993), was in and AN39Timl2 then cloned upstream of AN39Timl0, AN28Timl2, (a gift pSP64. The cyb2-targeting sequence was amplified from pSM4 from B. Glick, University of Chicago, IL)by PCR using appro Chicago, in the yeast GAL-Timl2 strain, the priate primers. For complementation cassette was subcloned whole cyb2AN28Timl2 and cyb2AN39Timl2 into pRS316, which contains the promoter and terminator from pSP64 of yeast Tim 12. ITS-AN23 (C28S) Sue fusion was generated by PCR using a primer that amplified the truncated segment of Sue containing a Cys to Ser substitution at position 28 and contained the 32-40-aa segment of yeast TimlO as a 5' overhang. The fusion protein ITS-DHFR was amplified by PCR using as template the vector pQEl?-DHFR The primer at the 5' end contained the 32-40-aa segment (QIAGEN). of yeast TimlO as an overhang. The fusion protein DHFR-ITS was ampli fied by PCR using as template the vector pQEl?-DHFR (QIAGEN) with a segment of yeast Tim 10 primer that contained at the 3' end the 32-40-aa as an overhang. The fusion proteins ITS-F33A-DHFR and DHFR-ITS-F33A were cloned as ITS-DHFR and DHFR-ITS, respectively, but the primer con

Import inyeast mitochondria 35S-labeled precursor proteins were synthesized using the TNT SP6-coupled was im transcription/translation kit (Promega). The radioactive material in the presence of ug of wild-type yeast mitochondria ported in 50-100 2 mM ATP and 2.5 mM NADH for the indicated time points at 30?C. Mito in 1.2 M sorbitol and 20 mM Hepes, chondria were then resuspended or pH 7.4, followed by a treatmentwith 0.1 mg ml-1 proteinase K with without 1% (vol/vol) Triton X-100 to remove unimported material. Finally, in Laemmli sample bufferwith or without ?-Me as theywere resuspended indicated, analyzed by SDS-PAGE, and visualized by digital autoradiog raphy (Molecular Dynamics). BN analysis of imported precursor After import of radioactive 35S-labeled protein inwild-type mitochondria, buffer (50 mM the samples were solubilized in0.16% N-dodecylmaltoside 1 mM NaCl, pH 7.4, 2.5 mMMgCl2/ 10%glycerol, 20 mM Hepes/KOH, EDTA, and 1 mM PMSF) for 30 min on ice. The solubilized material was then g, which was separated by a 20-min centrifugation at 100,000 and loaded on 6-16% gel (Sch?gger gradient BN electrophoresis von Jagow, 1991 ; Sch?gger et al., 1994). The radioactive material was de For antibody depletion, the solubilized mate tected by autoradiography. incubated with antibodies against Mia40 for 20 min on ice and rial was for 20 min on ice. beads thenwith protein A-Sepharose (GE Healthcare) The unbound material was separated by centrifugation at 2,500 g for 2 min before loading on the gradient BN gel. Coimmunoprecipitation of imported precursor inmitochondria After import of radioactive 35S-labeled protein inwild-type mitochondria for 10 min at 30?C, the samples were treatedwith 0.1 mg ml-1 proteinase K in to remove unimported material. Mitochondria were then resuspended

tained the 32-40-aa segment of yeast TimlO with a Phe substitution to Ala at position 33, which refers to the amino acid residue of thewild-type were cloned inpSP64 for invitro transcription/ yeast Tim 10. All fusion inserts






"7 2009

10 mM Tris, pH 7.4, 0.5% Triton X-100, and lysis buffer (150 mM NaCI, then diluted 1% SDS) and boiled for 5 min at 95?C. The sample was 10 mM Tris, 20 times with immunoprecipitation buffer (150 mM NaCI, pH 7.4, and 0.5% Triton X-100) and incubated with antibodies against beads for 2 h at 4?C. The bound mate Mia40 and protein A-Sepharose three timeswith immunoprecipitation buffer rial on the beads was washed in Laemmli sample buffer. The immunoprecipitated and then resuspended under reducing and nonreducing SDS-PAGE and material was analyzed visualized by digital autoradiography. Protein purification Recombinant proteins were and

with C55

and human TIM9 and of the transient complex between human MIA40 human MIA40 and human TIM10 were obtained with the between HADDOCK program (Dom?nguez et al., 2003), combining mutagenesis the modeling, Indeed, we constrained analysis with in silico docking. MIA40 (Banci et al., taking account of the mutational analysis data for and for the Tims (reported in this study) as well as the experi 2009) mental data that the N-terminal Cys of the Tims makes a disulfide pair

Sideris and Tokatlidis, of MIA40 (Milenkovic et al., 2007; The structures of MIA402S-s (PDB ID 2K3J) and fully reduced 2007). TIM9 and -10 were used as input. The fully reduced TIM9 and -10 struc tures were generated from the human TIM9-TIM10 complex structure


incubated with for30 min at 4?C and 21,000 g. The soluble fractionwas 1 ml of swollen glutathione beads per liter of culture overnight or for6 h at 4?C. Beads were washed with bufferA and then eluted with bufferA + 50 mM reduced glutathione. Then, 5 U/ml thrombinwas added and left to cut theGST tag overnight or for? h at 0?C. The cleaved fussed protein was isolated from theGST tag by gel filtration column with a Superdex75 (GE Healthcare). Ni-NTA beads pull-down assay was incubated with Ni-NTA beads Purified recombinant AN290Mia40SPS 1 ug of pure protein was used per 1 pi for 20 min at 4?C. (QIAGEN) of beads. The beads were washed with binding buffer (150 mM NaCI,

in bufferA + 50 mM imidazole. Then, the protein was eluted by bufferA + 300 mM imidazole. Eluted protein was further purified by anion on a MonoQ HR 5/5 column (GE Healthcare). exchange chromatography Cells carrying the pGEX constructs were harvested after induction with inbufferA 0.4 mM IPTG overnight at 18?C. Cells were then resuspended and sonicated for5 min at 50% amplification. Samples were centrifuged

AN39 TimlO as a GST fusion) constructs. Cells carrying the harvested after induction with plasmid were pET22AN290Mia40SPS in buffer A 0.4 mM IPTG for 3 h at 37?C. Cells were then resuspended (150 mM NaCI and 50 mM Tris HCI, pH 7.4) and sonicated for 5 min at 50% amplification. Samples were centrifuged for 30 min at 4?C and 21,000 g. Protein was purified from the soluble fraction. 15 mM imidaz ole was added and then loaded onto Ni-nitrilotriacetic acid (NTA) resin (QIAGEN) using 2.5 ml of resin per literof culture. Ni-NTA resin was

strainfrom AN30, pET22a (AN290Mia40SPS) or pGEX (wild type,


in the Escherichia

coli BL21(DE3)

and Cys29, respectively. Tyr21, Leu24, and Cys28 and Tyr22, Met25, in interaction were Cys55, MIA40 residues for engaged Corresponding Leu56, Met59, Phe72, Phe75, Phe91, and Met94, which were similarly combined with in vitro and in obtained from site-directed mutagenesis vivo assays A disulphide bond between Cys28 (Banci et al., 2009). in docking calculations. Structural models of the complexes between and MIA40 were obtained and yMia40 and between COX17 yCoxl7 with the HADDOCK program after the same aforementioned procedure. 2ZXT) were used as input in the human and yeast complexes, in theMia40 interaction for COX tively. The residues engaged

the oxidized TIM9 and -10 protomer structures and randomly opening in theMIA40 interaction for TIM9 two a helices. The residues engaged combined with in vitro and -10, obtained by site-directed mutagenesis and in vivo assays using the almost identical yeast homologues, were

the fully the twodisulphidebridges from (PDB ID 2BSK) by removing

TIM9 or Cys29 for for TIM10 and Cys55 ofMIA40 was also imposed

Mia40 (PDB ID MIA40 (PDB ID2K3J)and yeast The structures of human

respec 17 and with in vitro

obtained by site-directed mutagenesis combined yCoxl7, this study), were Leu48, Ile49, and in vivo assays (Banci et al., 2009; His52, and Cys45 and Phe50, Ile51, Tyr54, and Cys57, respectively. or Cys57 foryCoxl 7 and A disulphide bond between Cys45 forCOX17 or Cys298 of yMia40, Cys55 of MIA40 respectively, was also imposed in docking calculations. Miscellaneous

Triton 50 mM Tris, pH 7.4, 15 mM imidazole, 0.1% BSA, and 0.01% 35S-labeled proteins were produced using the TNT SP?-coupled treated transcription/translation kit. After the reaction, the lysates were with saturated (NH4)2S04 solution for 20 min in ice and centrifuged for 20 min at 16,000 g to precipitate the 35S-labeled protein and remove the in globins of the lysates. After precipitation, the pellet was resuspended X-100).

in the yeast strain GAL-Timl2 was performed as de Complementation In brief, the GAL-Timl2 strain scribed previously (Lionaki et al., 2008). was transformed with the pRS316 plasmid harboring the corresponding

under and cyb2AN39Timl2) genes (fusion constructs cyb2AN28Timl2 Tim 12 promoter. The transformed clones the control of the endogenous were grown inminimal medium containing 0.2% GAL and then shifted to 0.2% glucose for 36 h before the complementation test. The same dilution of cells was dropped on either SC or SG plates (minimal medium with 0.2% glucose or 0.2% GAL, respectively) and incubated at 30?C for4 d. SDS-PAGE was performed according to standard procedures. For the sep

tein, 20 ul of beads, and 175 ul of binding buffer) for2 h at the indicated temperature. After incubation, the beads were collected by centrifugation and washed three timeswith 180 ul of binding buffer. The bound material was resuspended in Laemmli sample buffer, separated by SDS-PAGE, and visualized by autoradiography.

denaturing buffer (8 M urea, 150 mM NaCI, 50 mM Tris HCI, pH 8.0, and 1 mM DTT) in a volume equal to the volume of the lysates before precipitation and incubated for 1 h at room temperature. The denatured in incubated with Ni-NTA beads equilibrated 35S-labeled proteins were (5 ul of denatured pro binding bufferwith or without AN290Mia40SPS

aration of proteins below 15 kD, Tris-Tricine SDS-PAGE was applied (Allen et al., 2003). Quantification of radioactive bands was performed using the Image Quant software (Molecular Dynamics), and error bars were generated by quantification of at least three independent experiments. In some figures, nonrelevant gel lanes were excised by digital treatment. to Western blots were performed on nitrocellulose membranes according standard procedures.

ITC was used to measure the association constant, enthalpy (AH), en tropy (AS), and free energy (AG) changes of TimlO variants binding to At least two independent measurements of the re AN290Mia40SPS. action were made at 25?C. All experiments were performed on VP-ITC microcalorimetry (MicroCal). Protein samples were extensively dialyzed

Online supplemental material and itsCys mutants with Mia40 Fig. SI shows the interaction of COX17 inmitochondria. Fig. S2 shows the interaction of yeast Coxl 7 and its Cys mutants with Mia40 inmitochondria. Fig. S3 shows the import controls as a percentage of the translation mix used for import. Fig. S4 shows the theo

against 50 mM KPi, pH 7.4, for 12 h at 4?C. To ensure full reduction of the proteins, they were incubated for 1 h with 10 mM TCEP (Tris

KPi, pH 7.4, and 1 mM TCEP at 4?C to make sure that the proteins were in the same buffer conditions. TimlO, AN30, and AN39 were used at was injected at 0.25 mM at 15-s inter 0.025 mM, and AN290Mia40SPS vals. The concentration of the proteins was determined cally using their respective extinction coefficients. spectrophotometri

at 4?C and then [2-carbxyethylJphosphine) dialyzed for1 hwith50 mM

Mia40 substrates. Online supplemental material prediction of ITS in able at We

retical modeling of the yeast Coxl 7 docking to the yeast Mia40 substrate binding cleft using HADDOCK. Fig. S5 shows the localization of ITS fusion constructs inwild-type mitochondria. Table SI lists the calculated param eters of the data-driven docking of substrates to Mia40. Table S2 shows the isavail

Helical wheel projection and HADDOCK

thank S. Karamanou (Foundation for Research and Technology Hellas, for help with ITC, N. Pfanner and A. Chacinska Heraklion, Crete, Greece) (Universityof Freiburg, Freiburg im Breisgau, Germany) for sharing unpub lished data, S. Makrogikas and P. Kritsiligkou forhelp with some mutants, and members of our group fordiscussions and comments.

Helical wheel representations were made using the helical wheel viewer for a helices from the University of Virginia inCharlottesville (http://cti Structural models

This work was supported by funds from the Institute ofMolecular Biol ogy and Biotechnology, Foundation for Research and Technology Hellas, the Universityof Crete, and the European Social Fund and national resources (to K. Tokatlidis), the European Network of Research Infrastructures for Providing



fon mitochondrial



* Sidenis

et ai.


Access and Technological Advancements in Bio-NMR (contract no. 026145 to I.Bertiniand L. Banci), and SPINE II (contract no. LSHG-CT-200?-031 220). D.P. Sideris and N. Petrakiswere supported by a PENED grant.

Mesecke, N., N. Terziyska, C. Kozany, F. Baumann, W. Neupert, K. Hell, and J.M. Herrmann. 2005. A disulfide relay system in the intermembrane space of mitochondria thatmediates protein import. Cell. 121:1059? 1069. doi: 10.1016/j.cell.2005.04.011 Milenkovic, D., K. Gabriel, B. Guiard, A. Schulze-Specking, N. Pfanner, and A. Chacinska. 2007. Biogenesis of the essential Tim9-Timl0 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptorMia40. J. Biol Chem. 282:22472 22480. doi: 10.1074/jbe.M703294200 Milenkovic, D., T. Ramming, J.M. M?ller, L.S. Wenz, N. Gebert, A. Schulze Specking, D. Stojanovski, S. Rospert, and A. Chacinska. 2009. Iden tificationof the signal directing Tim9 and TimlO into the intermembrane doi:10.1091/mbc space of mitochondria. Mol Biol. Cell. 20:2530-2539. .E08-11-1108 M?ller, J.M., D. Milenkovic, B. Guiard, N. Pfanner, and A. Chacinska. 2008. Precursor oxidation by Mia40 and Ervl promotes vectorial transportof proteins into the mitochondrial intermembrane space. Mol Biol. Cell. 19:226-236. doi:10.1091/mbc.E07-08-0814

25 May 2009 Submitted: Accepted: 19November2009

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