2 3 accepted - molecular and cellular biology

1

MCB02216-07 revised 1

2

The Assembly Pathway of the Mitochondrial Carrier Translocase 3

Involves four Preprotein Translocases 4

5

Karina Wagner,1,2 Natalia Gebert,1,2 Bernard Guiard,3 Katrin Brandner,1 Kaye 6

N.Truscott,1,4 Nils Wiedemann,1 Nikolaus Pfanner,1* and Peter Rehling1,5* 7

8

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-9

79104 Freiburg, Germany1; Fakultät für Biologie, Universität Freiburg, D-10

79104 Freiburg, Germany2; Centre de Génétique Moléculaire, CNRS, 91190 11

Gif-sur-Yvette, France3; Department of Biochemistry, La Trobe University, 12

Melbourne 3086, Australia4; Abteilung für Biochemie II, Universität Göttingen, 13

D-37073 Göttingen, Germany5 14

15

*Corresponding author. Mailing address for Nikolaus Pfanner: Institut für 16

Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 17

Freiburg, Germany. Phone: 49-761-203-5224. Fax: 49-761-203-5261. E-mail: 18

[email protected]. Mailing addresses for Peter 19

Rehling: Abteilung für Biochemie II, Universität Göttingen, Heinrich-Düker-20

Weg 12, D-37073 Göttingen, Germany. Phone: 49-551-39-5947. Fax: 49-551-21

39-5979. [email protected]. 22

23

Running title: Mitochondrial carrier translocase assembly 24

Keywords: Mitochondria, protein complex assembly, Tim protein, inner 25

membrane 26

ACCEPTED

Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.02216-07 MCB Accepts, published online ahead of print on 5 May 2008

on April 13, 2019 by guest

http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

2

ABSTRACT 1

2

The mitochondrial inner membrane contains preprotein translocases that 3

mediate insertion of hydrophobic proteins. Little is known about how the 4

individual components of these inner membrane preprotein translocases 5

combine to form multi-subunit complexes. We have analyzed the assembly 6

pathway of the three membrane-integral subunits Tim18, Tim22 and Tim54 of 7

the twin-pore carrier translocase. Tim54 displayed the most complex pathway 8

involving four preprotein translocases. The precursor is translocated across 9

the intermembrane space in a supercomplex of outer and inner membrane 10

translocases. The TIM10 complex, which translocates the precursor of Tim22 11

through the intermembrane space, functions in a new post-translocational 12

manner in case of Tim54, it is required for the integration of Tim54 into the 13

carrier translocase. Tim18, the function of which has been unknown so far, 14

stimulates integration of Tim54 into the carrier translocase. We show that the 15

carrier translocase is built via a modular process and that each subunit follows 16

a different assembly route. Membrane insertion and assembly into the 17

oligomeric complex are uncoupled for each precursor protein. We propose 18

that the mitochondrial assembly machinery has adapted to the needs of each 19

membrane-integral subunit and that the uncoupling of translocation and 20

oligomerization is an important principle to ensure continuous import and 21

assembly of protein complexes in a highly active membrane. 22

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

3

INTRODUCTION 1

2

The majority of mitochondrial proteins are nuclear-encoded and imported 3

into mitochondria through protein translocase complexes (6, 7, 17, 25, 29, 33, 4

42). The translocase of the outer membrane (TOM complex) is the general 5

entry gate for mitochondrial precursor proteins. Two translocases of the inner 6

membrane, the presequence translocase (TIM23 complex) and the twin-pore 7

carrier translocase (TIM22 complex), mediate signal-selective transport of 8

precursor proteins. While the TIM23 complex translocates the majority of 9

substrates into the matrix and inserts only a limited number of substrates into 10

the inner membrane (7, 10, 13, 17, 25, 26, 40), the TIM22 complex is 11

dedicated to the insertion of multispanning hydrophobic proteins into the inner 12

membrane, including a large number of metabolite carriers (7, 17, 25, 27, 29, 13

46). The TIM22 complex is a voltage-dependent 300 kDa complex with three 14

membrane-integral subunits, Tim18, Tim22, and Tim54. Tim22 forms the 15

voltage-sensitive channels of the twin-pore translocase (21, 30). Tim54 was 16

shown to play a role in the assembly of a protease complex (Yme1) of the 17

inner membrane, yet the molecular mechanism of its action has not been 18

elucidated (12). Thus, the molecular functions of Tim54 and Tim18 in the 19

TIM22 complex are unknown (6, 7, 17, 25, 29). 20

The precursors of metabolite carriers are not directly transferred from the 21

TOM complex to the TIM22 complex, but the TIM10 translocase complex of 22

the intermembrane space binds to the precursors and functions in a 23

chaperone-like manner to guide them through the aqueous space between 24

outer and inner membranes. The hexameric TIM10 translocase is formed by 25

the family of small Tim proteins. The soluble complex consists of three copies 26

of Tim10 and three copies of Tim9 (41). A fraction of small Tim proteins, 27

including Tim9, Tim10, and the homolog Tim12, associate with the TIM22 28

complex, forming a membrane-associated TIM10 chaperone. It is unknown 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

4

which Tim subunits mediate the contact between the TIM10 chaperone and 1

the membrane-integral portion of the TIM22 complex. 2

All subunits of the TIM22 complex are encoded in the nucleus and 3

synthesized in the cytosol. Initial analysis of the biogenesis of TIM22 subunits 4

have indicated that the precursors of Tim18 and Tim54 proteins utilize amino-5

terminal targeting signals and are imported via the presequence pathway 6

(TIM23 complex) (15, 16, 19, 22). In contrast, Tim22 lacks an amino-terminal 7

presequence and was proposed to be imported along the carrier pathway (22, 8

23, 35). 9

Proper assembly of inner mitochondrial membrane complexes is critical 10

for mitochondrial function since this membrane is pivotal for cellular energy 11

conversion through oxidative phosphorylation. It is crucial for the cell to 12

assemble the protein complexes that reside in the inner membrane in a 13

manner that excludes an uncontrolled flux of ions across the membrane in 14

order to prevent a breakdown of the electrochemical proton gradient. This is 15

especially true for protein complexes that contain channel-forming subunits 16

such as the preprotein translocase complexes. However, it is currently 17

unknown how the TIM complexes are assembled from newly imported 18

subunits and if the assembly to oligomeric complexes is coupled to the import 19

process. 20

We have dissected the in organello assembly pathways of all membrane-21

integral subunits of the TIM22 complex by establishing an efficient native 22

system. We show here that at different steps of the TIM22 complex 23

biogenesis pathway four translocases are involved. Remarkably, each 24

precursor follows a different assembly route. This involves a new post-25

translocational function of the TIM10 complex. Moreover, we obtained 26

evidence for a role of Tim18 in the assembly of the TIM22 complex and for a 27

cooperation of Tim54 with Tim10. We propose that the uncoupling of 28

membrane insertion of subunits from their subsequent oligomeric assembly 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

5

promotes an efficient biogenesis of translocase complexes. 1

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

6

MATERIALS AND METHODS 1

2

Yeast strains, growth conditions and isolation of mitochondria. All 3

yeast strains used in this study were derivatives of the S. cerevisiae strain 4

YPH499. Temperature sensitive alleles tim22-14 (YPH499 22-M4), tim54-11 5

(YPH-BG-54-1-1), tim54-16 (YPH-BG-54-1-6) and tim10-2 (YPH499 10-71-1) 6

were generated by error prone PCR (38). The tim18∆ strain was generated by 7

homologous recombination of a kanMX6 cassette into the TIM18 locus. Liquid 8

yeast cultures for the isolation of mitochondria were grown in YPG medium 9

(1% yeast extract, 2% bacto peptone, and 3% glycerol) at 30°C (tim22-14, 10

tim18∆, PRY19 (tim18ProtA) (30) and corresponding wild-type) or 24°C for all 11

other temperature-sensitive strains. Isolation of mitochondria was performed 12

essentially as described (24). 13

Import of radiolabeled precursor proteins. For in vitro transcription 14

and translation the open reading frames encoding Tim22, Tim18, and Tim54 15

were cloned into the pGEM4Z vector (Promega), either downstream of the 16

Sp6 promotor (Tim22 and Tim18) or the T7 promotor (Tim54) (22). 17

Radiolabeling of precursor proteins with [35S]methionine was performed with 18

the TNT Sp6 Quick Coupled Transcription/Translation System or the TNT 19

T7 Coupled Reticulocyte Lysate System (Promega). Import of radiolabeled 20

precursor proteins was performed essentially as described (44) and the 21

proteins or complexes were separated by SDS-PAGE or blue native 22

electrophoresis, respectively. Digital autoradiography was utilized for 23

detection. 24

Blue native electrophoresis. Mitochondria were solubilized under 25

non-denaturing conditions in digitonin-containing buffer (1% digitonin, 20 mM 26

Tris/HCl, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol and 1 mM PMSF) 27

for 30 min at 4°C and then centrifuged for 10 min at 16,000 x g. After addition 28

of 10 x loading dye (5% Coomassie brilliant blue G-250, 500 mM ε-amino n-29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

7

caproic acid in 100 mM Bis-Tris, pH 7.0) to the supernatant, the samples were 1

separated on a 6-16.5% gradient blue native gel (4, 5). For Western blot 2

analysis the proteins were transferred to a PVDF membrane and proteins of 3

interest were decorated with the appropriate antibodies, incubated with a 4

horseradish-conjugated secondary antibody and detected with the ECL 5

detection system (GE Healthcare). 6

Antibody-shift/antibody-depletion analysis. After import, mitochondria 7

were swollen in EM-buffer (1 mM EDTA, 10 mM MOPS-KOH, pH 7.2) and 8

antisera against Tim22 and Tim18 or BSA as a negative control were added 9

for binding to the protein complexes. Samples were incubated for 45 min on 10

ice and subsequently solubilized in digitonin-containing buffer. Binding of 11

antibodies to outer membrane protein complexes (Tom40 or porin) and inner 12

membrane protein complexes (Tim23 or Tim12) was performed during 13

solubilization of mitochondria in digitonin buffer by adding the appropriate 14

antiserum. Protein complexes were separated by blue native electrophoresis 15

and analyzed by autoradiography (4, 32). For antibody-depletion analysis, 16

solubilized mitochondria were incubated with purified lyophilized antibodies 17

(anti-Tim10 and anti-Atp20) for 5 min followed by the addition of Protein A-18

Sepharose and a 30 min incubation at 4°C. Protein A-Sepharose was 19

removed by centrifugation and the supernatant was analyzed by blue native 20

electrophoresis and digital autoradiography. 21

Pulse chase import experiment. Radiolabeled proteins were 22

imported for 5 min at 25°C into mitochondria (75 µg protein amount) in import 23

buffer (3% BSA, 250 mM sucrose, 80 mM KCl, 5 mM MgCl2, 2 mM KH2PO4, 5 24

mM methionine, 10 mM MOPS-KOH, pH 7.2) in the presence of 2 mM ATP 25

and NADH. To remove non-imported radiolabeled precursor proteins, 26

samples were centrifuged (10 min, 4°C, 16,000 x g) and mitochondria 27

resuspended in fresh import buffer. The chase reaction was carried out by 28

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

8

incubation for 20 min at different temperatures (25°C, 30°C and 37°C). After 1

the chase reaction, samples were subjected to blue native electrophoresis. 2

Carbonate extraction. After import, mitochondria were resuspended in 3

0.1 M sodium carbonate, pH 10.8 - 11.5, and incubated for 30 min on ice. 4

Pellet and supernatant fractions were separated by centrifugation (60 min, 5

4°C, 100,000 x g). Upon TCA-precipitation of supernatant and total, all 6

samples were subjected to SDS-PAGE. 7

Chemical crosslinking. Mitochondria (5 mg protein amount) from PRY19 8

cells (30) that contain Tim18ProtA were resuspended in SEM buffer (250 mM 9

sucrose, 1 mM EDTA, and 10 mM MOPS-KOH, pH 7.2) and incubated with 3-10

maleimidobenzoyl-N-hydroxy-succinimide-ester and disuccinimidyl glutarate 11

for 1 h at 16°C. Following quenching for 25 min on ice, samples were washed 12

with SEM buffer, resuspended in digitonin buffer (0.8% digitonin, 20 mM 13

Tris/HCl, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol and 1 mM PMSF) 14

and solubilized for 30 min at 4°C. After a clarifying spin IgG-Sepharose was 15

added to the supernatant and incubated for 90 min at 4°C. Bound proteins 16

were washed with digitonin buffer, eluted by treatment with TEV (tobacco etch 17

virus) protease for 90 min at 16°C, and separated by SDS-PAGE. For 18

combined import, chemical crosslinking and immunoprecipitation, 19

[35S]methionine labeled Tim54 was imported into Tim18ProtA mitochondria for 20

1 h at 35°C. After addition of ethylene glycol bis[succinimidylsuccinate] and 21

incubation for 90 min at 16°C the reactions were quenched for 10 min on ice. 22

Solubilization was done in digitonin buffer (0.8% digitonin, 20 mM Tris/HCl, pH 23

7.4, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol and 1 mM PMSF) for 15 min at 24

4°C. Mitochondrial extracts were incubated with IgG-Sepharose for 1h at 4°C 25

for complex purification. After washing in digitonin buffer, elution was 26

performed using SDS sample buffer lacking bromophenol blue (10 min at 27

room temperature). Upon heating to 95°C for 5 min and dilution with sample 28

buffer that contained 0.5% (w/v) Triton X-100 but lacked bromophenol blue 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

9

and SDS, samples were subjected to immunoprecipitation with anti-Tim9, 1

anti-Tim10 or anti-Tim12 antibodies covalently coupled to Protein A-2

Sepharose and incubated for 1 h at room temperature. Protein A-Sepharose 3

was sedimented by centrifugation and bound proteins were analyzed by SDS-4

PAGE and digital autoradiography. 5

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

10

RESULTS 1

2

Assembly of Tim18, Tim22 and Tim54 into the carrier translocase. To 3

analyze the biogenesis of the TIM22 complex we synthesized and 4

radiolabeled the precursor of the core component Tim22 in vitro. The 5

precursor was imported into isolated and energized yeast mitochondria and 6

non-imported precursor was removed by subsequent treatment of the 7

mitochondria with proteinase K. The mitochondria were reisolated and lysed 8

with the mild detergent digitonin under conditions that maintain the integrity of 9

the TIM22 complex, as well as that of the other mitochondrial preprotein 10

translocases (1, 22, 30, 43). Upon separation of the protein complexes by 11

blue native electrophoresis, a small amount of radiolabeled Tim22 was found 12

in the mature 300 kDa TIM22 complex (Fig. 1A, lanes 1-6). In addition, the 13

precursor of Tim22 accumulated in two low molecular weight forms on the 14

native gels. Formation of these forms required a membrane potential (∆ψ) 15

across the inner membrane (Fig. 1A, lanes 2-6 versus lane 1). As outlined 16

below, these forms likely represent monomeric and dimeric forms of Tim22 17

inserted into the inner membrane and are referred to as Tim22m and Tim22d 18

(Fig. 1A). 19

We generated temperature-conditional yeast mutants of TIM22 by error 20

prone PCR. The tim22-14 mutant strain was selected. The strain was inhibited 21

for growth at 37°C on non-fermentable medium (see Fig. S1A in the 22

supplemental material). Upon growth at permissive temperature, mitochondria 23

were isolated from tim22-14 and wild-type cells and analyzed for steady-state 24

protein levels. The amount of Tim22 was moderately reduced in tim22-14 25

mitochondria, while other subunits of the TIM22 complex and further 26

mitochondrial proteins were present in levels close to that of wild-type 27

mitochondria (see Fig. S1B in the supplemental material). Remarkably, the 28

mutant mitochondria showed a strongly increased efficiency of integration of 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

11

imported Tim22 into the mature TIM22 complex (Fig. 1A, lanes 8-12). 1

Concomitantly, the amounts of Tim22m and Tim22d were reduced, raising the 2

possibility that the low molecular weight forms may represent assembly 3

intermediates of Tim22, which are consumed to form the mature 300 kDa 4

complex (Fig. 1A, lanes 8-12). 5

We asked if the tim22-14 mutation lead to a special phenotype of an 6

increased assembly of the TIM22 complex and therefore imported the 7

radiolabeled precursors of the two other membrane-integral subunits of the 8

complex, Tim54 and Tim18. Both proteins assembled into the 300 kDa TIM22 9

complex of wild-type mitochondria in the presence of a ∆ψ (Fig. 1B and 1C, 10

lanes 2-6). In tim22-14 mutant mitochondria, however, the integration of 11

Tim54 as well as Tim18 into the TIM22 complex was strongly inhibited (Fig. 12

1B and 1C, lanes 7-11). To exclude indirect defects of the tim22-14 mutant 13

mitochondria on the presequence pathway, we imported the precursor of the 14

subunit β of the F1Fo-ATP synthase (F1β). The preprotein was imported with 15

similar efficiency in wild-type and mutant mitochondria (see Fig. S1C in the 16

supplemental material). Moreover, assessment of the membrane potential by 17

fluorescence quenching revealed that the tim22-14 mitochondria generated a 18

membrane potential that was only slightly lower than that of wild-type 19

mitochondria, excluding that the severe inhibition of the assembly of Tim54 20

and Tim18 was due to a dissipation of the ∆ψ (see Fig. S1D in the 21

supplemental material). Thus, tim22-14 mutant mitochondria show a 22

differential effect on the assembly of the TIM22 complex. While the integration 23

of the precursor of Tim22 into the complex is considerably enhanced, the 24

precursors of Tim54 and Tim18 are inhibited in assembly into the mutant 25

complex. 26

We thus screened for TIM54 temperature-conditional yeast mutants in 27

order to address the function of Tim54 in the TIM22 assembly process. We 28

selected the mutants tim54-11 and tim54-16, which were inhibited for growth 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

12

at 37°C on non-fermentable medium (see Fig. S2A in the supplemental 1

material). Upon growth at permissive temperature, the protein levels of 2

selected mitochondrial proteins were analyzed in isolated mitochondria. The 3

levels of Tim54 were decreased in the mutant mitochondria, while other 4

subunits of the TIM22 complex and further mitochondrial proteins were 5

present in wild-type amounts (see Fig. S2B in the supplemental material). The 6

tim54 mutant mitochondria were not impaired in the import of presequence-7

containing proteins, as shown with the matrix-targeted model preprotein 8

b2(167)∆DHFR (see Fig. S2C in the supplemental material). The membrane 9

potential of the tim54 mutant mitochondria was comparable to that of wild-type 10

mitochondria (see Fig. S2D in the supplemental material). Assembly of the 11

precursor of Tim22 into the TIM22 complex of both mutant mitochondria was 12

blocked (Fig. 1D, lanes 5-7 and 9-11). Strikingly, assembly of Tim54 into the 13

tim54 mutant mitochondria was significantly increased in comparison to its 14

assembly into wild-type mitochondria (Fig. 1E). The precursor of Tim18 did 15

not assemble into the TIM22 complex in tim54 mutant mitochondria (Fig. 1F, 16

lanes 5-7 and 9-11), similar to the observation made with the precursor of 17

Tim22. Thus, the tim54 mutant mitochondria yielded an assembly pattern that 18

was complementary to the pattern observed for tim22 mutant mitochondria. 19

Assembly of the wild-type precursor (Tim54 and Tim22, respectively) was 20

strongly enhanced in mitochondria containing a mutant version of this protein, 21

while assembly of the other two precursor proteins was inhibited. 22

To study if these findings may point to a general principle, we generated a 23

mutant of the third membrane subunit of the TIM22 complex, i.e. tim18∆ 24

mitochondria. In tim18∆ mitochondria the assembly of imported Tim18 into the 25

300 kDa TIM22 complex was indeed significantly increased compared to wild-26

type mitochondria (Fig. 1I). The precursor of Tim22 efficiently assembled into 27

the 250 kDa TIM22’ complex of tim18∆ mutant mitochondria (Fig. 1G, lanes 7-28

11) (Due to the lack of Tim18, the TIM22’ complex of tim18∆ mitochondria 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

13

migrates faster on blue native gels (16, 19)). Interestingly, when the precursor 1

of Tim54 was imported into tim18∆ mitochondria the assembly of Tim54 into 2

the 250 kDa TIM22’ complex was strongly decreased (Fig. 1H), suggesting 3

that Tim18 is involved in the assembly pathway of Tim54. 4

The precursor of Tim18 was also found in a low molecular weight form 5

that was formed in a ∆ψ-dependent manner (Fig. 1C, lanes 2-6 versus lane 6

1). In tim18∆ mitochondria, the amount of the low molecular weight form was 7

reduced while the formation of the mature TIM22 complex proceeded faster 8

(Fig. 1I), suggesting that this form may represent an intermediate on the 9

assembly pathway of Tim18. (In the case of Tim54, we also observed a low 10

molecular weight precursor form on the blue native gels. However, this form 11

was only partially affected by a dissipation of ∆ψ (Fig. 1B, 1E and 1H) and a 12

large fraction of this form was sensitive to externally added protease (see 13

below, Fig. 3A). Thus, the low molecular weight form of Tim54 likely does not 14

represent one defined species on the assembly pathway but is probably 15

formed from precursors at different stages of their import pathway.) 16

Taken together, we have established a native assay to monitor the 17

assembly of the three membrane-integral subunits of the TIM22 complex. The 18

assay revealed possible assembly intermediates for the precursors of Tim22 19

and Tim18 and a role of Tim18 in the assembly of Tim54. Strikingly, each of 20

the mutant mitochondria assembles the imported wild-type precursor, which 21

corresponds to the mutant subunit, with high efficiency while the assembly of 22

other wild-type subunits is impaired. 23

Assembly of Tim22 occurs via low molecular weight intermediate 24

forms. We used antibody-shift blue native electrophoresis (14, 38, 43) to 25

characterize the two low molecular weight forms of the Tim22 assembly 26

pathway, Tim22m and Tim22d. The radiolabeled precursor of Tim22 was 27

imported into mitochondria and surface-bound precursors were removed by 28

protease treatment. The mitochondria were subjected to swelling to rupture 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

14

the outer membrane and permit access of antibodies to the intermembrane 1

space and inner membrane. Upon addition of antibodies directed against a C-2

terminal epitope of Tim22 both small species were efficiently shifted (Fig. 2A, 3

lanes 2-4). However, the mature TIM22 complex was not shifted by the 4

antibodies (Fig. 2A, lanes 2-4), indicating that in the fully assembled complex 5

the epitope of Tim22 was not accessible from the intermembrane space while 6

it remained accessible in the two low molecular weight species. 7

The blue native gel mobility of Tim22m correlates with that of a monomer 8

of Tim22. When mitochondria were lysed in SDS-buffer prior to blue native 9

separation, all of Tim22 co-migrated with Tim22m (data not shown). Tim22d 10

may represent a homodimer or a heterodimer of Tim22. According to the gel 11

mobility, Tim18 would be a possible partner protein in a heterodimer (below a 12

range of 100 kDa, the blue native mobility of membrane proteins is slower in 13

comparison to that of the soluble 66 kDa marker protein (30, 31, 38)). 14

Antibodies directed against Tim18 shifted the mature TIM22 complex in a 15

dose-dependent manner but did not alter the mobility of the low molecular 16

weight species of Tim22 (Fig. 2A, lanes 5-7). Since the epitope of Tim18 was 17

exposed in the 300 kDa TIM22 complex, it is unlikely that the epitope would 18

be not accessible in a small complex. Together with the observation that 19

Tim22d was still formed in tim18∆ mitochondria (Fig. 1G), we conclude that 20

Tim18 is not a constituent of Tim22d. To probe for the possible presence of 21

Tim10 in Tim22d, we used an antibody-depletion assay. After import of Tim22, 22

solubilized mitochondria were mixed with anti-Tim10 antibodies, followed by 23

depletion using Protein A-Sepharose. The mature 300 kDa TIM22 complex 24

was depleted whereas Tim22d was not affected in comparison to control 25

antibodies (see Fig. S3A in the supplemental material), suggesting that Tim10 26

or the TIM10 chaperone complex were not present in Tim22d. Currently, it 27

cannot be excluded that other proteins are associated with Tim22 in Tim22d, 28

however, the available results are also compatible with a homodimer of 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

15

Tim22. 1

In order to determine if Tim22m and Tim22d represented intermediates on 2

the assembly pathway of Tim22, a pulse chase analysis was performed. The 3

precursor of Tim22 was imported into mitochondria for a short time such that 4

the two low molecular weight species were formed, but only a small amount of 5

the mature TIM22 complex (Fig. 2B, lane 4). The mitochondria were 6

reisolated and incubated at increasing temperatures. During this chase, 7

Tim22 assembled into the TIM22 complex in a temperature-dependent 8

manner and the amounts of small Tim22 species concomitantly decreased 9

(Fig. 2B, lanes 1-3). We conclude that Tim22m and Tim22d represent 10

intermediates of the Tim22 biogenesis pathway, explaining the reduced 11

amounts of the low molecular weight species when the integration of Tim22 12

into the TIM22 complex was accelerated in tim22-14 mitochondria (Fig. 1A). 13

The precursor of Tim22 is transported to the inner membrane via the 14

carrier pathway (18, 22, 23, 36). We used tim10-2 mutant mitochondria that 15

are impaired in the TIM10 chaperone complex of the intermembrane space (2, 16

8, 38). The cells were grown at permissive temperature, mitochondria were 17

isolated and subjected to a short heat shock. As expected, translocation of the 18

precursor of Tim22 to a protease-protected location was impaired upon loss of 19

Tim10 function in mitochondria (Fig. 2C) (18, 36). Blue native electrophoresis 20

analysis of Tim22 import into tim10-2 mitochondria revealed a strong inhibition 21

of formation of the two low molecular weight species as well as of the mature 22

TIM22 complex (Fig. 2D). To exclude that the lack of Tim22 assembly into the 23

TIM22 complex was due to dissociation of the pre-existing TIM22 complex 24

under the conditions applied, we performed a Western blot analysis of the 25

temperature-shifted mitochondria. The TIM22 complex remained stable under 26

these conditions (Fig. 2D, lanes 10, 12 and 14). In contrast, only small 27

amounts of the soluble TIM10 complex were observed (Fig. 2D, lane 14 28

versus lane 13). Thus, formation of the low molecular weight intermediates of 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

16

Tim22 requires a functional TIM10 complex. Moreover, the formation of the 1

intermediates requires the inner membrane potential ∆ψ (Fig. 1A and 2D). 2

The ∆ψ is required for the insertion of precursors into the inner membrane 3

after their ∆ψ-independent transfer across the intermembrane space (21, 30), 4

suggesting that the low molecular weight intermediates represent inner 5

membrane-inserted forms of the precursor. (The formation of Tim22d strongly 6

depends on the presence of a ∆ψ, while a fraction of the monomeric Tim22m 7

can also be observed in the absence of a ∆ψ. Tim22m likely includes two 8

monomeric species, a ∆ψ-dependent one that is inserted into the inner 9

membrane and a ∆ψ-independent one that is protected against externally 10

added proteinase K but not yet inserted into the inner membrane; the latter 11

intermediate has also been found for other precursors using the carrier 12

pathway (30, 31, 38)). In order to obtain further evidence that the low 13

molecular weight intermediates of Tim22 were integrated into the inner 14

membrane, we performed a treatment at alkaline pH to extract soluble and 15

peripheral membrane proteins. We used tim54-16 mitochondria, where the 16

imported radiolabeled Tim22 was only present in Tim22m and Tim22d but not 17

in the TIM22 complex (Fig. 1D, lanes 9-11), to selectively analyze the low 18

molecular weight intermediates. The majority of imported [35S]Tim22 was not 19

extracted at alkaline pH and thus behaved like the integral membrane proteins 20

Tom70 and preexisting Tim22, while Tim10 was found in the supernatant (see 21

Fig. S3B in the supplemental material). Taken together with the ∆ψ-22

dependence, these results indicate that the low molecular weight forms of 23

Tim22 are inserted into the membrane. 24

The mutants of Tim54 revealed a differentiation between Tim22m and 25

Tim22d. The formation of Tim22d but not of Tim22m was markedly decreased 26

in the tim54-11 mutant mitochondria (Fig. 1D, lanes 5-7). Thus, Tim22m is 27

formed despite the mutation in Tim54, while the generation of Tim22d 28

depends on the function of Tim54. These results suggest that the precursor of 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

17

Tim22 is inserted into the inner membrane as a monomeric form and then 1

converted to Tim22d in a Tim54-dependent manner. 2

Tim54 forms a TOM-TIM supercomplex during import. To analyze 3

early steps in the import of Tim54, mitochondria were incubated with the 4

radiolabeled precursor and separated by blue native electrophoresis without 5

treating the mitochondria with protease. We found a large protein complex 6

containing the precursor of Tim54 in addition to the TIM22 complex (Fig. 3A, 7

lanes 2-6). This complex was fully sensitive to a treatment with protease (Fig. 8

3A, lanes 8-12), indicating that the complex was exposed on the mitochondrial 9

surface, in contrast to the mature TIM22 complex. Surprisingly, the formation 10

of this complex strictly depended on a ∆ψ across the inner membrane (Fig. 11

3A, lanes 2-6 versus lane 1), raising the possibility that the precursor of Tim54 12

was accumulated in a two-membrane spanning fashion in a TOM-TIM 13

supercomplex. 14

A TOM-TIM supercomplex of a preprotein in transit has so far been 15

reported for arrested model preproteins, which contain a stably folded 16

passenger protein (3, 4, 11, 28, 34, 45). The large Tim54 complex would 17

represent the first TOM-TIM intermediate of an authentic preprotein that is 18

visualized by blue native electrophoresis. Thus, rigorous controls were 19

required to demonstrate the two-membrane accumulation of the precursor of 20

Tim54. Firstly, we analyzed if the Tim54-containing complex contained the 21

TOM complex of the outer membrane by using antibody-shift blue native 22

electrophoresis. Antibodies directed against Tom40, the central channel-23

forming subunit of the TOM complex (see Fig. S3C in the supplemental 24

material), quantitatively shifted the complex (Fig. 3B, lane 2) while antibodies 25

against porin did not (Fig. 3B, lane 3). In a control reaction, we imported the 26

receptor Tom22 into mitochondria. Tom22 assembled into the TOM complex 27

and was efficiently shifted with anti-Tom40 antibodies (Fig. 3B, lane 5). The 28

size of the Tim54-containing complex, however, was larger than that of the 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

18

TOM complex (Fig. 3B, lane 1 versus 4). We conclude that the precursor of 1

Tim54 was accumulated in a TOM-containing complex that apparently 2

contained additional components. 3

Import of Tim54 into mitochondria occurs through the presequence 4

translocase (22, 23). In agreement with this, in conditional tim50-1 mutant 5

mitochondria (1) the import of Tim54 was decreased compared to wild-type 6

mitochondria (Fig. 3C). The level of the large Tim54-containing complex was 7

strongly reduced in the mutant mitochondria (Fig. 3D, lanes 4 and 5). Thus, 8

functional Tim50 is critical for the formation of the Tim54-containing complex. 9

Together with the strict ∆ψ-dependence, we conclude that an active inner 10

membrane is required to form this large complex and thus the precursor of 11

Tim54 is likely accumulated in a two-membrane spanning fashion in a TOM-12

TIM supercomplex. This supercomplex may contain either the TIM23 complex 13

or the TIM22 complex. To directly probe for the presence of TIM complexes, 14

we used specific antibodies. Antibodies against Tim10 and Tim12 removed 15

the TIM22 complex as expected but did not influence the supercomplex, while 16

antibodies against Tim23 affected the supercomplex (Fig. 3E). We conclude 17

that the precursor of Tim54 was accumulated in a TOM-TIM23 supercomplex. 18

Assembly of Tim54 depends on the TIM10 complex. The TIM10 19

translocase complex is critical for the transport of proteins with internal 20

targeting signals from the TOM complex to the TIM22 translocase (8, 18, 23, 21

36, 38). For a few preproteins with amino-terminal targeting signals, a 22

dependence on the soluble TIM translocases of the intermembrane space 23

was also reported (23, 37). When we imported Tim54 into tim10-2 mutant 24

mitochondria, assembly of Tim54 into the TIM22 complex was severely 25

affected (Fig. 4A, lanes 4, 5, 10 and 11). However, the TOM-TIM 26

supercomplex of Tim54 was efficiently formed (Fig. 4A, lanes 4 and 5). This 27

indicated that the block of Tim54 biogenesis in tim10-2 mitochondria occurred 28

after the ∆ψ-dependent translocation of Tim54 into mitochondria. To test this, 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

19

we compared the Tim54 import efficiency between wild-type and tim10-2 1

mitochondria by treatment with proteinase K and analysis by SDS-PAGE. 2

Tim54 was transported to a protease-protected location in tim10-2 3

mitochondria with an efficiency that was close to that of wild-type 4

mitochondria (Fig. 4B). Tim54 imported into tim10-2 mitochondria was 5

resistant to extraction at alkaline pH like an integral membrane protein (Fig. 6

4C), indicating that the precursor was inserted into the membrane. Since the 7

TIM22 complex remained stable in tim10-2 mitochondria (Fig. 2D), we 8

concluded that the assembly of Tim54 into the TIM22 complex was selectively 9

affected by inactivation of the TIM10 complex at a post-membrane insertion 10

stage. As a control we analyzed the assembly of Tim18 in wild-type and 11

tim10-2 mitochondria. Tim18 was imported and assembled independently of 12

Tim10 (Fig. 4D). In summary, while the transport of Tim54 into the inner 13

membrane is not affected by inactivation of the TIM10 complex, assembly of 14

Tim54 into the carrier translocase depends on the TIM10 complex. This 15

mutant defect is selective for Tim54, since Tim18 biogenesis is not affected. 16

We asked if the TIM10 complex indirectly affected the assembly of Tim54 17

or if TIM10 and Tim54 interacted with each other in organello. Mitochondria 18

containing a Protein A-tagged version of Tim18 (30) were subjected to 19

chemical crosslinking. The TIM22 complex was isolated by IgG affinity 20

chromatography and analyzed by SDS-PAGE. Antibodies directed against 21

Tim54, as well as antibodies directed against Tim10 decorated a band of 22

about 65 kDa (Fig. 5A, lanes 2 and 4), suggesting that Tim54 may be in 23

proximity to Tim10. To obtain further evidence we imported radiolabeled 24

Tim54 into Tim18ProtA mitochondria. Upon purification of the TIM22 complex, 25

bound proteins were eluted under denaturing conditions and subjected to 26

immunoprecipitation with antibodies against Tim9, Tim10 or Tim12. The 65-27

kDa crosslinking product was specifically precipitated with antibodies against 28

Tim10, but not with antibodies against Tim9 or Tim12. Thus, Tim10 is in close 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

20

proximity to Tim54 in the TIM22 complex, supporting the view that Tim10 1

plays a direct role in the assembly of Tim54. 2

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

21

DISCUSSION 1

2

We developed an in organello assay to monitor the biogenesis and 3

assembly pathway of each membrane-integral subunit of the mitochondrial 4

carrier translocase. We show that each precursor follows a different assembly 5

pathway. Remarkably, up to four preprotein translocase complexes are 6

required for import and assembly. Thus, for the precursor of Tim54, all known 7

protein import translocases of mitochondria, TOM, TIM23, TIM10, and TIM22, 8

are required, representing one of the most complex protein assembly 9

pathways of mitochondria known to date. 10

Despite the diversity of the import routes, an important principle emerged, 11

that is an uncoupling of translocation into the inner membrane from the 12

subsequent assembly to the oligomeric complex. The mitochondrial inner 13

membrane has to translocate entire polypeptide chains and assemble 14

membrane-spanning proteins into oligomeric complexes. Each of these 15

processes bears the danger of disturbing the barrier of the inner membrane 16

that is essential to maintain the electrochemical proton gradient. The proton 17

gradient is the main driving force for the bulk of cellular ATP synthesis, as well 18

as for the import of precursor proteins, and a leakage of the inner membrane 19

would be deleterious. Since membrane insertion of an individual unfolded 20

polypeptide chain and the assembly of different proteins into a large complex 21

are mechanistically quite different processes, a combination of both 22

processes would substantially increase the risk of a non-specific ion leakage. 23

We thus analyzed how translocation and assembly are separated for the 24

individual precursor proteins. 25

(i) The precursor of Tim22 is transferred from outer to inner membranes 26

by the TIM10 translocase complex of the intermembrane space and forms low 27

molecular weight assembly intermediates in the inner membrane before the 28

subsequent integration into the 300 kDa TIM22 complex. The ∆ψ-dependent 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

22

insertion into the inner membrane takes place at the level of the low molecular 1

weight intermediates and is thus uncoupled from the assembly into the TIM22 2

complex. Electrophysiological analysis of the TIM22 complex in comparison to 3

reconstituted Tim22 alone indeed showed that the Tim22 channel is highly 4

active in the TIM22 complex and performs rapid gating transitions in response 5

to a membrane potential and targeting peptides, while Tim22 alone shows a 6

significantly lower activity (30), suggesting that the uncoupling of membrane 7

insertion and oligomerization reduces the risk of an unspecific leakage of ions. 8

(ii) The precursor of Tim54 is transferred across the intermembrane space by 9

a different mechanism. A cooperation of TOM and TIM23 complexes leads to 10

a two-membrane spanning preprotein-TOM-TIM23 supercomplex. The ∆ψ-11

dependent insertion of Tim54 into the inner membrane is required for 12

formation of this supercomplex and occurs before the assembly of Tim54 into 13

the TIM22 complex. The separation of translocation and assembly was 14

directly shown by an unexpected new function of the TIM10 translocase 15

complex of the intermembrane space. The TIM10 complex is not required for 16

the translocation of Tim54 from outer to inner membranes but for its 17

subsequent incorporation into the 300 kDa complex. (iii) The precursor of 18

Tim18, which is also imported by the presequence route (9, 16, 19), is 19

inserted into the inner membrane in a ∆ψ-dependent manner, forming a low 20

molecular weight form. This form is consumed when the assembly of Tim18 21

into the TIM22 complex is enhanced, indicating that the low molecular weight 22

form represents the inner membrane-inserted intermediate form. Interestingly, 23

the TIM10 translocase is neither needed for translocation nor assembly of 24

Tim18. 25

A recent study on the biogenesis of a subunit of the F1F0 ATP synthase of 26

Escherichia coli revealed that oligomerization was not a prerequisite for 27

membrane insertion of the protein (20) and the sorting and assembly 28

machinery of the mitochondrial outer membrane (SAM complex) performs the 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

23

tasks of membrane insertion and assembly in consecutive steps (43). We 1

propose that an uncoupling of membrane insertion and oligomeric assembly 2

may represent a general mechanism for the biogenesis of membrane protein 3

complexes. 4

The assembly of each wild-type precursor of a Tim protein into the TIM22 5

complex was strongly enhanced when mutant mitochondria were used that 6

were defective in exactly this subunit. The assembly of the other two subunits 7

was not enhanced in the mutant mitochondria but decreased in most cases. 8

This behavior was observed for each of the three membrane-integral 9

subunits. A related result was observed for one subunit of the TIM23 complex 10

(39), however, it could not be decided if this represented a unique case for 11

this subunit or a more general principle. The systematic analysis of all three 12

membrane subunits of the TIM22 complex now provides the basis to 13

formulate a general principle for the assembly of a hetero-oligomeric 14

membrane protein complex. We propose that a destabilization of the TIM22 15

complex in the mutant mitochondria facilitates the integration of individual 16

newly imported subunits. Under wild-type conditions, an incoming precursor 17

protein has to replace a pre-existing subunit or to associate with non-18

assembled pools of both other subunits that are likely small under wild-type 19

conditions. Thus, the efficiency of integration into the wild-type complex is 20

limited. When the TIM22 complex is labilized by a mutation of an individual 21

subunit, the incoming wild-type subunit can much more easily replace the pre-22

existing (mutant) subunit. Moreover, destabilization of the complex will 23

increase the pools of non-assembled subunits and thus the incoming wild-24

type subunit can rapidly associate with the other two subunits to form the 25

holo-translocase. 26

Our studies reveal evidence for the possible molecular functions of Tim18 27

and Tim54 in the TIM22 complex. Tim18 has been found as a stoichiometric 28

subunit of the TIM22 complex (16, 19), however, its role remained unclear. 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

24

While the precursor of Tim22 still assembled into a smaller TIM22 complex in 1

mitochondria lacking Tim18, the assembly of the precursor of Tim54 was 2

inhibited. The assembly of Tim54 was not blocked completely, consistent with 3

the viability of yeast cells lacking Tim18 (16, 19), but strongly retarded in the 4

in organello assay, suggesting that Tim18 may be involved in the efficient 5

integration of Tim54 into the TIM22 complex. Tim54 exposes a large domain 6

to the intermembrane space (15) and was thus an interesting candidate for 7

the docking site of small Tim proteins at the TIM22 complex, however, 8

experimental evidence has been lacking. We observed an efficient cross-9

linking of Tim54 to Tim10, suggesting that the link between intermembrane 10

space translocase and the membrane portion of the TIM22 complex is 11

mediated by Tim10 and Tim54. The contact of Tim54 to Tim10 is not only 12

needed for docking of the TIM10 complex to the TIM22 complex but also for 13

the incorporation of the Tim54 precursor into the TIM22 complex. Thus, the 14

small Tim protein of the intermembrane space is critical for the assembly of 15

the membrane-integral portion of the carrier translocase, suggesting that the 16

cooperation of Tim54 and Tim10 represents an important element for both 17

biogenesis and function of the TIM22 complex. 18

Taken together, each subunit of the twin-pore carrier translocase follows a 19

different assembly pathway. Along the assembly pathways distinct 20

intermediate complexes are formed and inserted into the inner membrane 21

before the association to the large complex. We speculate that a dimeric form 22

of the central pore-forming subunit Tim22 is the initial building block to which 23

Tim54 associates in a TIM10- and Tim18-dependent manner. Thus, the multi-24

step maturation pathway of the carrier translocase separates the steps of 25

membrane insertion and oligomeric assembly. 26

27

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

25

ACKNOWLEDGMENTS 1

We thank M. van der Laan, A. Chacinska, and C. Meisinger for discussion 2

and comments on the manuscript. This work was supported by the Deutsche 3

Forschungsgemeinschaft, the Sonderforschungsbereich 746, Gottfried 4

Wilhelm Leibniz Program, Max Planck Research Award, and the Fonds der 5

Chemischen Industrie. 6

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

26

REFERENCES 1

2

1. Chacinska, A., M. Lind, A. E. Frazier, J. Dudek, C. Meisinger, A. 3

Geissler, A. Sickmann, H. E. Meyer, K. N. Truscott, N. Pfanner, and P. 4

Rehling. 2005. Mitochondrial presequence translocase: switching 5

between TOM tethering and motor recruitment involves Tim21 and Tim17. 6

Cell 120:817-829. 7

2. Chacinska, A., S. Pfannschmidt, N. Wiedemann, V. Kozjak, L. K. 8

Sanjuan Szklarz, A. Schulze-Specking, K. N. Truscott, B. Guiard, C. 9

Meisinger, and N. Pfanner. 2004. Essential role of Mia40 in import and 10

assembly of mitochondrial intermembrane space proteins. EMBO J. 11

23:3735-3746. 12

3. Chacinska, A., P. Rehling, B. Guiard, A. E. Frazier, A. Schulze-13

Specking, N. Pfanner, W. Voos, and C. Meisinger. 2003. Mitochondrial 14

translocation contact sites: separation of dynamic and stabilizing elements 15

in formation of a TOM-TIM-preprotein supercomplex. EMBO J. 22:5370-16

5381. 17

4. Dekker, P. J. T., F. Martin, A. C. Maarse, U. Bömer, H. Müller, B. 18

Guiard, M. Meijer, J. Rassow, and N. Pfanner. 1997. The Tim core 19

complex defines the number of mitochondrial translocation contact sites 20

and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. 21

EMBO J. 16:5408-5419. 22

5. Dekker, P. J. T., H. Müller, J. Rassow, and N. Pfanner. 1996. 23

Characterization of the preprotein translocase of the outer mitochondrial 24

membrane by blue native electrophoresis. Biol. Chem. 377:535-538. 25

6. Dolezal, P., V. Likic, J. Tachezy, and T. Lithgow. 2006. Evolution of the 26

molecular machines for protein import into mitochondria. Science 27

313:314-318. 28

7. Endo, T., H. Yamamoto, and M. Esaki. 2003. Functional cooperation 29

and separation of translocators in protein import into mitochondria, the 30

double membrane bounded organelles. J. Cell Sci. 15:3259-3267 31

8. Frazier, A. E., A. Chacinska, K. N. Truscott, B. Guiard, N. Pfanner, 32

and P. Rehling. 2003. Mitochondria use different mechanisms for 33

transport of multispanning membrane proteins through the intermembrane 34

space. Mol. Cell. Biol. 23:7818-7828. 35

9. Geissler, A., A. Chacinska, K. N. Truscott, N. Wiedemann, K. 36

Brandner, A. Sickmann, H. E. Meyer, C. Meisinger, N. Pfanner, and 37

P. Rehling. 2002. The mitochondrial presequence translocase: an 38

essential role of Tim50 in directing preproteins to the import channel. Cell 39

111:507-518. 40

10. Glick, B. S., A. Brandt, K. Cunningham, S. Müller, L. R. Hallberg, and 41

G. Schatz. 1992. Cytochromes c1 and b2 are sorted to the intermembrane 42

space of yeast mitochondria by a stop-transfer mechanism. Cell 69:809-43

822. 44

11. Horst, M., S. Hilfiker-Rothenfluh, W. Oppliger, and G. Schatz. 1995. 45

Dynamic interaction of the protein translocation systems in the inner and 46

outer membranes of yeast mitochondria. EMBO J. 14:2293-2297. 47

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

27

12. Hwang, D. K., S. M. Claypool, D. Leuenberger, H. L. Tienson, and C. 1

M. Koehler. 2007. Tim54p connects inner membrane assembly and 2

proteolytic pathways in the mitochondrion. J. Cell Biol. 178:1161-1175. 3

13. Jensen, R. E., and A. E. Johnson. 2001. Opening the door to 4

mitochondrial protein import. Nat. Struct. Biol. 8:1008-1010. 5

14. Johnston, A. J., J. Hoogenraad, D. A. Dougan, K. N. Truscott, M. 6

Yano, M. Mori, N. J. Hoogenraad, and M. T. Ryan. 2002. Insertion and 7

assembly of human tom7 into the preprotein translocase complex of the 8

outer mitochondrial membrane. J. Biol. Chem. 277:42197-42204. 9

15. Kerscher, O., J. Holder, M. Srinivasan, R. S. Leung, and R. E. Jensen. 10

1997. The Tim54p-Tim22p complex mediates insertion of proteins into the 11

mitochondrial inner membrane. J. Cell Biol. 139:1663-1675. 12

16. Kerscher, O., N. B. Sepuri, and R. E. Jensen. 2000. Tim18p is a new 13

component of theTim54p-Tim22p translocon in the mitochondrial inner 14

membrane. Mol. Biol. Cell 11:103-116. 15

17. Koehler, C. M. 2004. New developments in mitochondrial assembly. 16

Annu. Rev. Cell Dev. Biol. 20:309-335. 17

18. Koehler, C. M., E. Jarosch, K. Tokatlids, K. Schmid, R. J. Schweyen, 18

and G. Schatz. 1998. Import of mitochondrial carriers mediated by 19

essential proteins of the intermembrane space. Science 279:369-373. 20

19. Koehler, C. M., M. P. Murphy, N. A. Bally, D. Leuenberger, W. 21

Oppliger, L. Dolfini, T. Junne, G. Schatz, and E. Or. 2000. Tim18p, a 22

new subunit of the Tim22 complex that mediates insertion of imported 23

proteins into the yeast mitochondrial inner membrane. Mol. Cell. Biol. 24

20:1187-1193. 25

20. Kol, S., B. R. Turrell, J. de Keyzer, M. van der Laan, N. Nouwen, and 26

A. J. M. Driessen. 2006. YidC-mediated membrane insertion of assembly 27

mutants of subunit c of the F1F0 ATPase. J. Biol. Chem. 281:29762-29878 28

21. Kovermann, P., K. N. Truscott, B. Guiard, P. Rehling, N. B. Sepuri, H. 29

Müller, R. E. Jensen, R. Wagner, and N. Pfanner. 2002. Tim22, the 30

essential core of the mitochondrial protein insertion complex, forms a 31

voltage-activated and signal-gated channel. Mol. Cell 9:363-373. 32

22. Kurz, M., H. Martin, J. Rassow, N. Pfanner, and M. T. Ryan. 1999. 33

Biogenesis of Tim proteins of the mitochondrial carrier import 34

pathway:differential targeting mechanisms and crossing over with the 35

main import pathway. Mol. Biol. Cell 10:2461-2474. 36

23. Leuenberger, D., N. A. Bally, G. Schatz, and C. M. Koehler. 1999. 37

Different import pathways through the mitochondrial intermembrane 38

space for inner membrane proteins. EMBO J. 18:4816-4822. 39

24. Meisinger, C., N. Pfanner, and K. N. Truscott. 2006. Isolation of yeast 40

mitochondria. Methods Mol. Biol. 313:33-39. 41

25. Neupert, W., and J. M. Herrmann. 2007. Translocation of Proteins into 42

Mitochondria. Annu. Rev. Cell Dev. Biol. 76:723-749. 43

26. Oka, T., and K. Mihara. 2005. A railroad switch in mitochondrial protein 44

import. Mol. Cell 18:145-146. 45

27. Palmieri, L., F. M. Lasorsa, A. Vozza, G. Agrimi, G. Fiermonte, M. J. 46

Runswick, J. E. Walker, and F. Palmieri. 2000. Identification and 47

functions of new transporters in yeast mitochondria. Biochim. Biophys. 48

Acta 1459:363-369. 49

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

28

28. Rassow, J., B. Guiard, U. Wienhues, V. Herzog, F. U. Hartl, and W. 1

Neupert. 1989. Translocation arrest by reversible folding of a precursor 2

protein imported into mitochondria. A means to quantitate translocation 3

contact sites. J. Cell Biol. 109:1421-1428. 4

29. Rehling, P., K. Brandner, and N. Pfanner. 2004. Mitochondrial import 5

and the twin-pore translocase. Nat. Rev. Mol. Cell Biol. 5:519-530. 6

30. Rehling, P., K. Model, K. Brandner, P. Kovermann, A. Sickmann, H. E. 7

Meyer, W. Kühbrandt, R. Wagner, K. N. Truscott, and N. Pfanner. 8

2003. Protein insertion into the mitochondrial inner membrane by a twin-9

pore translocase. Science 299:1747-1751. 10

31. Ryan, M. T., H. Müller, and N. Pfanner. 1999. Functional staging of 11

ADP/ATP carrier translocation across the outer mitochondrial membrane. 12

J. Biol. Chem. 274:20619-20627. 13

32. Ryan, M. T., W. Voos, and N. Pfanner. 2001. Assaying protein import 14

into mitochondria. Methods Cell. Biol. 65:189-215. 15

33. Schnell, D. J., and D. N. Hebert. 2003. Protein translocons. 16

Multifunctional mediators of protein translocation across membranes. Cell 17

112:491-505. 18

34. Schülke, N., N. B. Sepuri, and D. Pain. 1997. In vivo zippering of inner 19

and outer mitochondrial membranes by a stable translocation 20

intermediate. Proc. Natl. Acad. Sci. USA 94:7314-7319. 21

35. Sirrenberg, C., M. F. Bauer, B. Guiard, W. Neupert, and M. Brunner M. 22

1996. Import of carrier proteins into the mitochondrial inner membrane 23

mediated by Tim22. Nature 384:582-585. 24

36. Sirrenberg, C., M. Endres, H. Fölsch, R. A. Stuart, W. Neupert, and M. 25

Brunner. 1998. Carrier protein import into mitochondria mediated by the 26

intermembrane space proteins Tim10/Mrs11 and Tim12/Mrs5. Nature 27

391:912-915. 28

37. Tatsuta, T., K. Model, and T. Langer. 2005. Formation of Membrane-29

bound Ring Complexes by Prohibitins in Mitochondria. Mol. Biol. Cell 30

16:248-259 31

38. Truscott, K. N., N. Wiedemann, P. Rehling, H. Müller, C. Meisinger, N. 32

Pfanner, and B. Guiard. 2002. Mitochondrial import of the ADP/ATP 33

carrier: the essential TIM complex of the intermembrane space is required 34

for precursor release from the TOM complex. Mol. Cell. Biol. 22:7780-35

7789. 36

39. Van der Laan, M., M. Meinecke, J. Dudek, D. P. Hutu, M. Lind, I. 37

Perschil, B. Guiard, R. Wagner, N. Pfanner, and P. Rehling P. 2007. 38

Motor-free mitochondrial presequence translocase drives membrane 39

integration of preproteins. Nat. Cell Biol. 9:1152-1159. 40

40. Van der Laan, M., M. Rissler, and P. Rehling. 2006. Mitochondrial 41

preprotein translocases as dynamic molecular machines. FEMS Yeast 42

Res. 6:849-861. 43

41. Webb, C. T., M. A. Gorman, M. Lazarou, M. T. Ryan, and J. M. Gulbis. 44

2006. Crystal structure of the mitochondrial chaperone TIM9-10 reveals a 45

six-bladed alpha-propeller. Mol. Cell 21:123-133. 46

42. Wickner, W., and R. Schekman. 2005. Protein translocation across 47

biological membranes. Science 310:1452-1456. 48

43. Wiedemann N, V. Kozjak, A. Chacinska, B. Schönfisch, S. Rospert, 49

M. T. Ryan, N. Pfanner, and C. Meisinger. 2003. Machinery for protein 50

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

29

sorting and assembly in the mitochondrial outer membrane. Nature 1

424:565-571. 2

44. Wiedemann, N., N. Pfanner, and P. Rehling. 2006. Import of precursor 3

proteins into isolated yeast mitochondria. Methods Mol. Biol. 313:373-4

384. 5

45. Wienhues, U., K. Becker, M. Schleyer, B. Guiard, M. Tropschug, A. L. 6

Horwich, N. Pfanner, and W. Neupert. 1991. Protein folding causes an 7

arrest of preprotein translocation into mitochondria in vivo. J. Cell Biol. 8

115:1601-1609. 9

46. Zara, V., A. Ferramosca, L. Capobianco, K. M. Baltz, O. Randel, J. 10

Rassow, F. Palmieri, and P. Papatheodorou. 2007. Biogenesis of yeast 11

dicarboxylate carrier: the carrier signature facilitates translocation across 12

the mitochondrial outer membrane. J. Cell Sci. 120:4099-4106. 13

14

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

30

FIGURE LEGENDS 1

2

FIG. 1. Membrane-integral components of the TIM22 complex assemble via 3

different intermediates. 4

(A) 35S-radiolabeled Tim22 (B) Tim54, and (C) Tim18 were imported into 5

isolated tim22-14 mitochondria at temperatures of 16°C to 25°C in the 6

presence or absence of ∆ψ and subsequently treated with 50 µg/ml 7

proteinase K (Prot. K). After solubilization in digitonin buffer, samples were 8

subjected to blue native electrophoresis and analyzed by digital 9

autoradiography. Import of (D) Tim22, (E) Tim54, and (F) Tim18 into isolated 10

tim54-11 and tim54-16 mitochondria and subsequent sample analysis was 11

carried out as described for (A - C). (G) Tim22, (H) Tim54, and (I) Tim18 were 12

imported into tim18∆ mitochondria as described above. Arrowhead, low 13

molecular weight form of Tim54; asterisk, low molecular weight intermediate 14

of Tim18; WT, wild-type. 15

16

FIG. 2. Tim22 assembly occurs through low molecular weight intermediates. 17

(A) Antibody-shift analysis of imported Tim22 in wild-type mitochondria. After 18

swelling of mitochondria, complexes were shifted by incubation with 19

increasing amounts of antisera against Tim22 and Tim18. After solubilization 20

with digitonin buffer protein complexes were separated by blue native 21

electrophoresis and analyzed by digital autoradiography. (B) Pulse chase 22

analysis of Tim22 assembly. 35S-labeled Tim22 was imported into 23

mitochondria as indicated in the scheme and samples were analyzed by blue 24

native electrophoresis and digital autoradiography. (C) Radiolabeled Tim22 25

was imported into wild-type (WT) and tim10-2 mitochondria in the presence or 26

absence of a ∆ψ and treated with 50 µg/ml proteinase K. Import was carried 27

out at 25°C after a 15 min pre-incubation of mitochondria at 37°C. Samples 28

were subjected to SDS-PAGE and analyzed by digital autoradiography. (D) 29

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

31

Import of Tim22 precursor protein was done as described in C and samples 1

were analyzed by blue native electrophoresis and digital autoradiography (left 2

panel). Isolated wild-type and tim10-2 mitochondria were incubated at 37°C 3

for 15 min prior to solubilization in digitonin buffer and separation of 4

complexes by blue native electrophoresis. Western blot analysis was 5

performed with the indicated antiserum (right panel). 6

7

FIG. 3. Tim54 forms a TOM-TIM intermediate. 8

(A) Assembly of radiolabeled Tim54 in wild-type mitochondria. After import of 9

Tim54, samples were left untreated or treated with 50 µg/ml proteinase K, 10

solubilzed in digitonin buffer and then analyzed by blue native electrophoresis 11

and digital autoradiography. (B) After import of radiolabeled Tim54, 12

complexes were shifted with antisera against Tom40 and Porin, or incubated 13

with BSA. As a control, Tom22 was imported and shifted as described for 14

Tim54. Analysis was carried out by blue native electrophoresis and digital 15

autoradiography. (C) Radiolabeled Tim54 was imported into isolated wild-type 16

and tim50-1 mitochondria in the presence or absence of ∆ψ. After treatment 17

with proteinase K proteins were separated by SDS-PAGE and visualized by 18

digital autoradiography. (D) 35S-labeled Tim54 was imported into wild-type 19

(WT) and tim50-1 mitochondria. After treatment with digitonin-containing 20

buffer, complexes were separated by blue native electrophoresis and 21

subjected to digital autoradiography. (E) Tim54 was imported into wild-type 22

mitochondria and subjected to antibody-shift/depletion analysis as described 23

in Materials and Methods. Samples were analyzed by blue native 24

electrophoresis and digital autoradiography. Arrowhead, low molecular weight 25

form of Tim54. 26

27

FIG. 4. Tim54 assembly is dependent on the TIM10 complex. 28

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

32

(A) Assembly of Tim54 in tim10-2 mutant mitochondria. Radiolabeled Tim54 1

was imported into isolated wild-type (WT) and tim10-2 mitochondria in the 2

presence or absence of a ∆ψ after a 15 min pre-incubation at 37°C. Where 3

indicated mitochondria were treated with proteinase K. Complexes were 4

separated by blue native electrophoresis and visualized by digital 5

autoradiography. (B) 35S-labeled Tim54 was imported as described in A, 6

including treatment with proteinase K. Samples were analyzed by SDS-PAGE 7

and digital autoradiography. (C) Radiolabeled Tim54 was imported. The 8

mitochondria were treated with proteinase K and subjected to treatment with 9

carbonate. Total (T), pellet (P) and supernatant (S) were analyzed by SDS-10

PAGE and digital autoradiography ([35S]Tim54) or immunodecoration (Tom70, 11

Mge1). (D) Radiolabeled Tim18 was imported into wild-type and tim10-2 12

mitochondria as described in A. Arrowhead, low molecular weight form of 13

Tim54; asterisk, low molecular weight intermediate of Tim18. 14

15

FIG. 5. Tim54 and Tim10 interact at the carrier translocase. 16

(A) Tim18ProtA mitochondria were subjected to chemical crosslinking as 17

described in Materials and Methods. The TIM22 complex was purified by IgG-18

Sepharose chromatography. After washing and elution, proteins were 19

separated by SDS-PAGE and analyzed by immunodecoration with antibodies 20

against Tim54 and Tim10. Circle, unspecific crossreaction of anti-Tim10. (B) 21

After import of radiolabeled Tim54 into Tim18ProtA mitochondria, proteins were 22

crosslinked as described in Materials and Methods and the TIM22 complex 23

was purified with IgG-Sepharose. After washing and elution of the proteins, an 24

immunoprecipitation was performed with antibodies against Tim9, Tim10 and 25

Tim12. Samples were analyzed by SDS-PAGE and digital autoradiography. 26

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

A B C

60 10 20 30 45 60 10 20 6060 30 45min

∆ψ - + ++ ++ + +- + + +

tim22-14WT

[35S]Tim22

1 2 3 4 5 6 7 8 9 10 11 12

kDa

66

140

232

440

669

TIM22complex

Tim22d

Tim22m

60 5 15 30 45 60 5 15 606030 45min

∆ψ - + ++ ++ + + -+ + +

tim22-14WT

[35S]Tim18

TIM22complex

1 2 3 4 5 6 7 8 9 10 11 12

∗

kDa

66

140

232

669

440

60 5 15 30 45 60 5 15 606030 45min

∆ψ - + ++ ++ + + -+ + +

tim22-14WT

[35S]Tim54

kDa

66

140

232

440

669

TIM22complex

1 2 3 4 5 6 7 8 9 10 11 12

E[35S]Tim22

min

∆ψ

30

-

15

+

30

+

15

+

30

-

30

+

30

-

WT tim54-11 tim54-16

TIM22complex

+

5

+

5 5

+

15

+

30

+

kDa

66

140

232

440

669

1 2 3 4 5 6 7 8 9 10 11 12

Tim22d

Tim22m

D

TIM22complex

[35S]Tim18

3015 30 15 30 15 3030 30min

∆ψ -+ + ++ + -+-


66

140

232

440

669

kDa555+ + +

1 2 3 4 5 6 7 8 9 10 11 12

∗

F

1 2 3 4 5 6 7 8 9 10 11 12

TIM22complex

30min

∆ψ -

3015 30 30 1515 3030

-+ + -+ +++

[35S]Tim54


66

140

232

440

669

kDa5 5 5

+ + +

1 2 3 4 5 6 7 8 9 10 11 12

60 5 15 30 45 60 5 15 606030 45min

∆ψ - + ++ ++ + + -+ + +

WT tim18∆

[35S]Tim18

kDa

66

140

232

440

669

TIM22complex

∗

I

60 5 15 30 45 60 5 15 606030 45min

∆ψ - + ++ ++ + + -+ + +

tim18∆WT

[35S]Tim22

kDa

66

140

232

440

669

TIM22complex

TIM22complex

1 2 3 4 5 6 7 8 9 10 11 12

Tim22d

Tim22m

G H

60 5 15 30 45 60 5 15 606030 45min

∆ψ - + ++ ++ + + -+ + +

tim18∆WT

[35S]Tim54

kDa

66

140

232

440

669

TIM22complex

TIM22complex

1 2 3 4 5 6 7 8 9 10 11 12

Wagner et al. Figure 1

´ ´

ACCEPTED on April 13, 2019 by guest

http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


1 2 3 4

C tim10-2 WT

20 2 5 10 15 20 5 2 15 10 20 20 min

∆ψ - + + + + + + - + + + +

8 9 10 2 3 4 5 1 6 7 11 12

[35S]Tim22

B

25 30 37 25 °C

TIM22 complex

kDa

66

140

232

440

669

Tim22d

Tim22m

Reisolation of

mitochondria

Mitochondria + [35S]Tim22

5 min 25°C

Puls

e

Chase 20 min

A

D

30 20 20 10 10 30 30 30 min

∆ψ + + + - + + + -

WT tim10-2

[35S]Tim22

kDa

66

140

232

440

669

1 2 3 4 5 6 7 8

TIM10 complex

13 14 9 10 11 12

tim

10-2

WT

WT

tim

10-2

tim

10-2

WT

Anti-

Tim22

Anti-

Tim54

Anti-

Tim10

Tim22d

Tim22m

TIM22 complex

1 2 3 4 5 6 7

µl serum

TIM22 complex

Tim22d

Tim22m

5 10 1 10 1 5 BS

A

kDa

66

140

232

440

669

Anti-

Tim22

Anti-

Tim18

Antibody-shift

Mitochondria + [35S]Tim22

Swelling of mitochondria

10 min 25°C

ACCEPTED on A

pril 13, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

http://mcb.asm.org/


A C tim50-1 WT

20 5 10 15 20 5 15 20 20 min

∆ψ - + + + + + + + + -

10

4 5 6 1 2 3 7 8 9 10

[35S]Tim54

min 15 5 45 60

∆ψ + + + + +

30 60

-

10 11 12 1 2 3 4 5 6 7 8 9

60 5 30 45 15 60

- + + + + +

[35S]Tim54

+ Prot. K - Prot. K

kDa

66

140

232

440

669

TIM22 complex

TOM-TIM

B

BS

A

An

ti-T

om

40

An

ti-P

orin

BS

A

An

ti-T

om

40

An

ti-P

orin

[35S]Tim54 [35S]Tom22

1 2 3 4 5 6

kDa

TIM22 complex

TOM-TIM

66

140

232

440

669

TOM

D

kDa

66

140

232

440

669

tim50-1 WT

[35S]Tim54

30 15 15 30 30 30

- + + + + -

min

∆ψ

1 2 3 4 5 6

TIM22 complex

TOM-TIM

kDa

66

140

232

440

669

Pre

imm

un

e

An

ti-T

im1

0

An

ti-A

tp2

0

[35S]Tim54

TIM22 complex

TOM-TIM A

nti-T

im2

3

An

ti-T

im1

2

1 2 3 4 5

E

‣

‣

‣

‣

ACCEPTED on April 13, 2019 by guest

http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


A

B tim10-2 WT

20 2 5 10 15 20 5 2 15 10 20 20 min

∆ψ - + + + + + + - + + + +

10 11 12 1 2 3 4 5 6 7 8 9

[35S]Tim54

66

140

232

440

669

kDa 15 30 30 15 30 30 30 15 15 30 30 30 min

∆ψ - + + + + + + - - + + -

tim10-2 WT tim10-2 WT

- Prot. K + Prot. K

[35S]Tim54

10 11 12 1 2 3 4 5 6 7 8 9

TIM22 complex

TOM-TIM

C tim10-2 WT

1 2 3 4 5 6

[35S]Tim54

Tom70

Mge1

T P S T P S

66

140

232

440

669

kDa 30 15 30 15 30 30 15 30 15 30 30 30 min

∆ψ + + - + + - + - - + + +

tim10-2 WT tim10-2 WT

- Prot. K + Prot. K

[35S]Tim18

10 11 12 1 2 3 4 5 6 7 8 9

TIM22 complex

∗

D

‣

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


kDa

1 2 3 4 5

Crosslinker - +

Total

+ + +

An

ti-T

im9

An

ti-T

im1

0

An

ti-T

im1

2

66

45 36

29 24

20

14

Tim54 Tim54XL

B A

Tim10XL

o

Tim10

Anti-

Tim54

- + Crosslinker

66

45 36

29 24

20

14

- +

Anti-

Tim10

Tim54 Tim54XL

1 2 3 4

ACCEPTED


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

2 3 accepted - molecular and cellular biology

Documents