Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane

Accepted Manuscript Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane

Vincenzo Zara, Alessandra Ferramosca, Kathrin Günnewig, Sebastian Kreimendahl, Jan Schwichtenberg, Dina Sträter, Mahmut Çakar, Kerstin Emmrich, Patrick Guidato, Ferdinando Palmieri, Joachim Rassow PII: DOI: Reference:

S0022-2836(18)30642-9 doi:10.1016/j.jmb.2018.09.012 YJMBI 65880

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

15 June 2018 18 September 2018 18 September 2018

Please cite this article as: Vincenzo Zara, Alessandra Ferramosca, Kathrin Günnewig, Sebastian Kreimendahl, Jan Schwichtenberg, Dina Sträter, Mahmut Çakar, Kerstin Emmrich, Patrick Guidato, Ferdinando Palmieri, Joachim Rassow , Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane. Yjmbi (2018), doi:10.1016/j.jmb.2018.09.012

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ACCEPTED MANUSCRIPT Revised manuscript JMB_2018_253 Article

Mimivirus-encoded nucleotide translocator VMC1 targets the mitochondrial inner

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membrane

Vincenzo Zara 1,#, Alessandra Ferramosca 1,#, Kathrin Günnewig 2, Sebastian Kreimendahl 2,

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Jan Schwichtenberg 2 , Dina Sträter 2 , Mahmut Çakar 2 , Kerstin Emmrich 2 ,

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Patrick Guidato 2,*, Ferdinando Palmieri 3 , Joachim Rassow 2

1 - Department of Environmental and Biological Sciences and Technologies, University of

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Salento, 73100 Lecce, Italy

2 - Institute for Biochemistry and Pathobiochemistry, Ruhr-University Bochum,

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44780 Bochum, Germany

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3 - Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari ,

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70125 Bari, Italy

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*Present address: Leibniz Institut für Analytischen Wissenschaften, Dortmund, Germany

# Both authors contributed equally to this study

Correspondence to Joachim Rassow: Institute for Biochemistry and Pathobiochemistry, Ruhr-University Bochum, 44780 Bochum, Germany. [email protected] Phone +49 (0)234 32-29079; Fax +49 (0)234 32-14266

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ACCEPTED MANUSCRIPT Abstract Mimivirus (Acanthamoeba polyphaga mimivirus, APMV) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study we investigated the

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intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast

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Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial

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outer membrane protein Tom70 and translocates through the import channel formed by the -

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barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by NEthylmaleimide (NEM) caused a delay in translocation but not a complete occlusion. Cell

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viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species (ROS) were affected. Similar to

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endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific

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translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute

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nucleotides for the replication of the large DNA genome of the virus.

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Keywords: Mimivirus, VMC1, mitochondrial carrier family, mitochondria, Tom40.

Abbreviations used:

AAC, ADP/ATP carrier; APMV, Acanthamoeba polyphaga

mimivirus; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCFH-DA, 2′,7′Dichlorodihydrofluorescein diacetate; DHFR, dihydrofolate reductase; MCF, mitochondrial carrier family; NEM, N-Ethylmaleimide; TIM, translocase of the mitochondrial inner membrane; TOM, translocase of the mitochondrial outer membrane; VF, virus factory/viral factory; VMC1, Viral mitochondrial carrier 1 2

ACCEPTED MANUSCRIPT Introduction Mimivirus is a giant DNA virus, its fiber-covered icosahedral protein capsid has a diameter of 0.75 μm, its genome comprises 1.18 Mbp 1,2. It was identified in Acanthamoeba polyphaga, a protist widely distributed in soil and water. Several related viruses were identified in the same species, forming the family of Mimiviridae 3. To date, 979 genes have been identified in the

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mimivirus genome 2. These include several genes encoding tRNAs, aminoacyl-tRNA

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syntethases, or other proteins involved in protein biosynthesis 4,5, challenging a basic distinction between viral and cellular genomes. An almost complete translation apparatus was

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recently discovered in the related Tupanvirus 6. However, the large majority of the mimivirus

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genes have no known function and make up a ‘functional dark matter’ 7. Correspondingly, only a few Acanthamoeba polyphaga mimivirus proteins have been studied experimentally.

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About 10% of the genes are probably acquired by horizontal gene transfer from prokaryotic or eukaryotic organisms 2. A member of this group is the gene L276. It encodes a protein of 237

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residues (27,4 kDa) with obvious similarities to mitochondrial metabolite carriers

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(Supplemental data, Fig. 1). In a previous study 8, the protein was isolated and reconstituted in artificial membranes. The investigations revealed an activity in the transport of several

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nucleotides with a preference for the deoxyribonucleotides dATP and dTTP 8. Similar to the activities of related mitochondrial proteins, transport of substrates across the membranes was

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only possible in an exchange reaction: dATP was transported in exchange for ADP, dTTP, TTP, or UTP. The nucleotide composition of the mimivirus genome is 72% A and T residues1. Mimivirus may thus exploit the mitochondrion of its host by using its dATP and dTTP for the facilitated replication of its large A/T-rich genome, possibly in exchange for cytosolic ADP. Because of the similarities to mitochondrial metabolite transporters in structure and function, the protein was named viral mitochondrial carrier 1 (VMC1) 8. However, the localization of VMC1 in eukaryotic host cells has so far not been determined.

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ACCEPTED MANUSCRIPT Mitochondrial carrier proteins are structurally related proteins of the inner mitochondrial membrane (mitochondrial carrier family, MCF) encoded by genes of the solute carrier family 25 (SLC25) 9. In human tissues, 53 members of this family were identified 9,10,11. In their basic organisation, they share a scheme of three modules of similar size, each module comprising about 100 amino acid residues forming two membrane-spanning hydrophobic -

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helices that are connected by hydrophilic loops. Typically, each module contains a signature

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motif, P-X-[DE]-X-X-[KR] 12,13. The VMC1 fulfils all these criteria. However, MCF proteins

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are not necessarily mitochondrial proteins: human mitochondria contain the citrate carrier mCiC, but a closely related isoform of the protein, pmCiC, mediates a transport of citrate

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across the plasma membrane 14; HsPMP34, encoded by the human gene SLC25A17, is a transporter of FMN, FAD, coenzyme A and NAD+ and exclusively localized in peroxisomes ; a Ca2+-binding MCF protein was identified in the peroxisomes of rabbits 17; also yeast

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Ant1, a transporter of ATP, is a peroxisomal protein 18; yeast Ugo1 is clearly related to MCF

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proteins and targeted to mitochondria, but acts as an integral protein of the mitochondrial

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outer membrane 19,20. VMC1, due to its small size of only 237 residues, could similarly be targeted to an unusual membrane. Most carrier proteins contain more than 300 residues, for

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example 313 residues in the case of the ADP/ATP carrier of Neurospora crassa 21.

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The biogenesis of endogenous mitochondrial inner membrane carrier proteins shows a common pattern of targeting and translocation 13,22,23,24,25,26. Newly synthesized carrier proteins contain internal targeting sequences that are recognized by components of the translocase of the mitochondrial outer membrane (TOM complex). The main recognition site is provided by the outer membrane protein Tom70 27,28. The general import pore of the outer membrane is formed by the -barrel protein Tom40 29,30,31,32,33. Integration of MCF proteins into the inner membrane is mediated by the TIM22 complex and dependent on the

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ACCEPTED MANUSCRIPT mitochondrial membrane potential. A complex of small chaperone proteins (Tim9 and Tim10) is required for the transfer from the TOM to the TIM complex 13,22,23,24,25,26,24.

In this study, we expressed VMC1 in HeLa cells and in yeast and found that VMC1 is specifically targeted to mitochondria. Following passage through the outer membrane import

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channel formed by Tom40, VMC1 eventually targets the mitochondrial inner membrane.

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Expression of VMC1 in HeLa cells or in yeast did not cause any direct toxicity, indicating

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that VMC1 primarily acts as a virus-encoded mitochondrial nucleotide translocator.

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Results

Intracellular distribution of VMC1 in mammalian cells

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To investigate the localization of VMC1 in an intact mammalian cell, we expressed the

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protein in HeLa cells using the vector pTET zeo+ one (Fig. 1a). A (His)10-tag was added at the C-terminus for detection by specific antibodies. Antibodies directed against TRAP-1 (a

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mitochondrial member of the Hsp90 family) were used for comparison. We found that VMC1 showed a clear co-localization with TRAP-1 (Fig. 1a, upper panel). A co-localization was also

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observed with Mitotracker Orange, a fluorescent dye for membrane potential-dependent labelling of mitochondria (Fig. 1a, lower panel). Under these conditions, the mitochondrial membrane potential was obviously retained, indicating that VMC1 did not impair the function of the respiratory chain or the integrity of the mitochondrial membranes. Correspondingly, we did not observe any changes in the morphology of the mitochondria or any significant toxicity for the cells. Alterations of the mitochondrial membrane potential easily cause an increased production of reactive oxygen species (ROS). We therefore used the ROS-sensitive fluorescent dye 5

ACCEPTED MANUSCRIPT 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) to monitor possible changes in the ROS content of the cells 35 (Fig. 1b). By addition of the uncoupling reagent CCCP (carbonyl cyanide m-chlorophenylhydrazone) together with Antimycin A (an inhibitor of respiratory chain complex III), the ROS production of HeLa cells was increased by a factor of 2 to 5, in agreement with data of previous studies 35. In contrast, we did not observe any significant

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effects by expression of VMC1 or the mitochondrial outer membrane protein Tom20 (Fig. 1c

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and d). Different from many other virus-encoded proteins 36,37,38, VMC1 appears to target

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mitochondria but to keep essential mitochondrial and cellular functions intact.

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To re-examine the mitochondrial localization of VMC1, we used a cell-free system (Fig. 2). VMC1, devoid of additional moieties, was synthesized in reticulocyte lysate in the presence

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of 35S-methionine and found to be completely degraded by low concentrations of proteinase K (Fig. 2a). To test for import into mitochondria, the radiolabelled protein was incubated with

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freshly isolated rat liver mitochondria at 25°C (Fig. 2b). After different times, samples

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received 75 g/ml proteinase K to degrade all accessible VMC1, and the mitochondria were reisolated by centrifugation. Within 10 min about 4% of the VMC1 was imported into a

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protease-protected location inside the mitochondria. Under the same conditions, AAC (ADP/ATP carrier, an abundant member of the mitochondrial carrier family 21) was imported

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with an efficiency of about 5.5%. VMC1 is thus targeted to mammalian mitochondria with an efficiency comparable to authentic mitochondrial proteins.

Mitochondrial targeting of VMC1 To investigate the interactions of VMC1 with mitochondria in more detail, we used yeast mitochondria as a well-established model system 39. We expressed VMC1 fused to the amino terminus of enhanced green fluorescent protein (EGFP) in yeast using the vector pUG36 and labelled the mitochondria by addition of Mitotracker Orange. The fusion protein co-localized 6

ACCEPTED MANUSCRIPT with the mitochondria of the cells (Fig. 3a). In agreement with the distribution of VMC1 in HeLa cells, a co-localization with other membrane systems was not observed. The mitochondria retained their membrane potential in the presence of VMV1, as shown by the efficient staining with the membrane potential-sensitive dye. The observations confirm the assumption of Monné et al. 8 that VMC1 is specifically targeted to mitochondria. To test for

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toxicity, we expressed VMC1 containing a His-tag and inoculated agar plates with

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suspensions of the yeast cells at different concentrations (Fig. 3b and c). The growth of the

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cells closely resembled the pattern observed with cells expressing the AAC (ADP/ATP carrier, Fig. 3c, upper panel vs. lower panel). Similar as in the HeLa cells, VMC1 seems to

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lack a significant toxicity.

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We then tested 35S-labelled VMC1 for import into isolated yeast mitochondria, again using the AAC 21 for comparison (Fig. 4a). After import, the mitochondria were treated with

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proteinase K, reisolated, and the relative amounts of radiolabelled protein imported into a

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protease-protected location were determined. Within 12 min at 25°C, about 6% of the added VMC1 were imported, vs. about 10% of AAC. To investigate the efficiency of targeting, both

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the reticulocyte lysates and the mitochondria were depleted of ATP by incubation with apyrase, mixed, and subsequently incubated at 25°C (Fig. 4b). Under these conditions, carrier

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proteins accumulate at the mitochondrial outer surface 40,41. Within 2 min nearly 20% of the AAC and 12% of the VMC1 were found associated with the mitochondria (Fig. 4b). The experiments show that VMC1 resembles the AAC in rapid binding to the mitochondria, import into the organelles proceeds at a considerably slower rate.

Newly synthesized mitochondrial proteins are initially directed to Tom70, Tom20 or Tom22, three receptor proteins of the mitochondrial outer membrane TOM complex 23,25,42. We compared the relative binding efficiencies of VMC1 to the isolated cytosolic domains of these 7

ACCEPTED MANUSCRIPT proteins (Fig. 4c). The experiments showed that VMC1 preferentially targets Tom70: In our assays, < 4% and < 2% bound to Tom20 or Tom22, respectively, but about 14% of VMC1 bound to Tom70. The pattern differed from the data obtained with Su9-DHFR (a protein containing an amino-terminal presequence for import into the mitochondrial matrix, 43), but

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showed similarities to the distribution found for the AAC.

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To determine the localization of the radiolabelled VMC1 inside the mitochondria, the

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organellar membranes were fragmented by sonication and the membrane vesicles were separated by sucrose density centrifugation (Fig. 4d). The fractions were analysed by SDS-

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PAGE and western blotting, Tom40 (outer membrane) and Tim23 (inner membrane) were used as marker proteins. VMC1 showed a clear co-fractionation with the inner membrane

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protein Tim23.

To characterize the import pathway of radiolabelled VMC1 in comparison to an authentic

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mitochondrial protein, we used blue native gel polyacrylamide gel electrophoresis (BN-

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PAGE, 44; Fig. 4e). The method allows a separation of different complexes of radiolabelled carrier proteins in distinct steps of translocation across the mitochondrial membranes 40,41,45,46.

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The pattern obtained with VMC1 resembled the pattern of other carrier proteins, confirming that VMC1 followed the same import pathway (Fig. 4e). The final step in the biogenesis of

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mitochondrial carrier proteins is the formation of functional proteins embedded in the mitochondrial inner membrane. Mature carrier proteins form complexes of about 90 kDa which can be visualized in BN-PAGE 45,46,47,48,49,50. A complex of this type was also detectable for the VMC1 (Fig. 4e, lanes 3 and 6). Integration into the inner membrane is strictly dependent on the mitochondrial membrane potential  (import stage V, 46,51). Correspondingly, the formation of the 90 kDa complex was completely blocked after dissipation of the membrane potential by valinomycin (Fig. 4e, lanes 2 vs. lane 3 and lane 5 vs. lane 6). 8

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Translocation through the Tom40 import channel Mitochondrial proteins are imported via Tom40, a -barrel protein of the outer membrane TOM complex. To investigate the participation of Tom40 in the import of VMC1, we used a competition assay: proteins that are attached to a stably folded domain can be arrested in the

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lumen of Tom40, the import of other proteins is thereby impaired 52,53,54. For this purpose, we

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used a hybrid protein comprising the 47 N-terminal residues of yeast cytochrome b2 fused to mouse dihydrofolate reductase (b2-DHFR, 54). The folding state of the DHFR can be

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stabilized by the ligand methotrexate 54. We expressed b2-DHFR in Escherichia coli and used

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the isolated hybrid protein to pre-incubate isolated yeast mitochondria with increasing amounts in the presence of methotrexate. The mitochondria were reisolated and subsequently

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incubated for 10 min with radiolabelled VMC1 (Fig. 4f). For comparison, parallel samples were incubated with radiolabelled yeast Ugo1 (a mitochondrial outer membrane protein, ), F1 (the -subunit of the mitochondrial ATP synthase, 56), and AAC 21. The proteins

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19,20,55

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showed striking differences: By pre-incubation with b2-DHFR, the amounts of imported F1 were reduced by about 67%. In contrast, the uptake of Ugo1 was reduced by only 7%. The

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AAC showed an intermediate sensitivity, b2-DHFR inhibited the import by 40%. The differences are in agreement with observations from previous studies showing that

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presequence-targeted precursor proteins such as b2-DHFR primarily block the inner membrane TIM23 complex, leaving a significant higher number of Tom40 channels open for the uptake of proteins such as the AAC which translocate independently of Tim23 53,57. Ugo1 is imported independently of both Tim23 and Tom40 58. However, as shown in Fig. 4f, b2DHFR inhibited the import of VMC1 to a similar degree (by about 32%) as the import of the AAC, in agreement with the notion that uptake of VMC1 is mediated by Tom40 but independent of the inner membrane TIM23 complex. 9

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There is compelling evidence that mitochondrial carrier proteins traverse the Tom40 channel in a loop structure 34,41,59. Hence, two polypeptide chains pass the narrow channel at the same time. Electrophysiological data on the Tom40 pore of yeast have indicated an average pore size of 22 Å 29. Considering that the diameter of an -helical polypeptide is about 12 Å, it

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should easily be possible to achieve a complete occlusion of the import pore, for example by

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modification of some of the amino acid side chains of the Tom40 polypeptide chain.

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The -barrel of yeast Tom40 is formed by 19 -strands 32. Interestingly, -strands 7, 17, 18 and 19 each contain a cysteine residue within a similar distance from the outer opening (C165

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of 7, C326 of 17, C341 of 18 and C355 of 19, 32; Fig. 5a and 5b). We therefore tested if a modification of these residues by reaction with N-Ethylmaleimide (NEM) may be sufficient

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to close the import channel. The mitochondria were pre-treated with trypsin and valinomycin to exclude interactions with outer membrane receptor sites or effects of the membrane

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potential, NEM was applied at a concentration of 2 mM for 20 min at 25°C. Under these

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conditions, the pre-incubation with NEM reduced the amounts of imported AAC by about 33%, higher concentrations of NEM did not cause a more pronounced inhibition (Fig. 5c).

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The import of dicarboxylate carrier (DIC, 60) was inhibited by about 31% (Fig. 5d). VMC1 was even less affected, its import was reduced by only 21% (Fig. 5d).

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A derivatization of the cysteine sulfhydryl groups by NEM is obviously not sufficient to close the Tom40 -barrel for import of VMC1 or other carrier proteins. Even during uptake of carrier protein loop structures, the capacity of the import pore is not operating at its limits.

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Discussion

In a previous study 8, VMC1 had been reconstituted in artificial membranes and shown to act as an efficient translocator of several nucleotides and thus to resemble the members of the

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mitochondrial metabolite carrier family both in structure and function. In this study, (i) we investigated if VMC1 indeed is a mitochondrial protein, (ii) we tested for possible toxic

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effects upon expression in intact cells, and (iii), following the confirmation of the

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mitochondrial location, we investigated the pathway of VMC1 during passage across the

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mitochondrial outer membrane.

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VMC1 shows by its primary structure that it is related to the mitochondrial metabolite carrier proteins. However, several members of this protein family are no mitochondrial proteins but

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specifically localized in peroxisomes or even in the plasma membrane 14,15,16,17,18. For VMC1,

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our experiments both with intact cells and with isolated mitochondria, clearly confirm a mitochondrial location. With regard to the possible function of VMC1 in the context of mimivirus replication, it is particularly remarkable that VMC1 eventually targets the

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mitochondrial inner membrane. In this location, VMC1 is able to mediate an exchange of

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nucleotides between the mitochondrial matrix and the cytosol. The substrate specificities of VMC1 could easily permit an export of mitochondrial deoxyribonucleotides to support the replication of the huge DNA genome of the mimivirus, using cytosolic ADP for exchange 8. According to the biophysical data of Monné et al. 8, VMC1 is highly specific for nucleotides. This observation is corroborated by our finding that VMC1 does not interfere with the mitochondrial membrane potential. In all our experiments, we did not find any evidence of a VMC1-related proton leakage, an increase in the mitochondrial production of reactive oxygen species, or of a significant VMC1-mediated toxicity. We therefore suggest that VMC1 indeed 11

ACCEPTED MANUSCRIPT acts as a specific nucleotide translocator in the mitochondrial inner membrane to support the replication of the mimivirus genome. The acquisition of the VMC1 gene seems to reflect the extraordinary demands on the availability of nucleotides for the synthesis of the huge mimivirus genome. Nearly all currently known DNA viruses carry out replication and transcription either entirely or partially within host nuclei. Remarkably, the infection cycle of

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mimivirus occurs exclusively in the host cytoplasm 61. The sites of viral replication are

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located in distinct cytoplasmic viral factories (VF) that are surrounded by membranes and form distinct compartments 62,63,64. Interestingly, 3-dimensional reconstructions of the

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replication centers show that the viral factories contain mitochondria 63. The close vicinity of

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viral replication and mitochondria should facilitate both the exchange of nucleotides and the

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delivery of mimivirus-encoded VMC1 to the mitochondria.

Only recently, it was discovered that the mimivirus genome contains segments that

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correspond to parts of the 5S, 16S and 23S ribosomal RNAs encoded by the Acanthamoeba’s

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mitogenome 65,66. The order of the viral segments is similar to the order of the corresponding genes in the mitochondrial genome, and it was considered that Mimiviridae may have

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mitochondria-like ancestors 66. Is the VMC1 a remnant of a mitochondrial prehistory of the Mimiviridae? Unfortunately, the evolutional origin of the VMC1 gene is not known, a close

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homologue in a eukaryotic genome was not identified 8. Mitochondrial carrier proteins are usually encoded by nuclear genes, however, also some bacterial genes were reported to encode proteins of this family 67. The origin of the VMC1 remains to be established.

Import of newly synthesized VMC1 into mitochondria seems to follow entirely the pathways of endogenous members of the mitochondrial metabolite carrier family. We did not find any deviation from the pathways of other carrier proteins, nor mimivirus-specific peculiarities. The interactions of VMC1 with the mitochondrial outer and inner membrane protein import 12

ACCEPTED MANUSCRIPT machinery closely resemble the typical pattern observed with other members of the carrier family 13,22,25,26. In our study, we were able to take advantage of the availability of new data on the molecular structure of Tom40 that were only recently resolved 32,33. The Tom40 barrel is formed by 19 -strands and has an irregular, roughly elliptical shape, with a shortest diameter of 11 Å and a longest diameter of 32 Å 33. The -strands of yeast Tom40 contain 4

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cysteine residues of similar distance from the outer opening of the channel 32. The array of 4

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cysteines seems to be non-essential as indicated by the fact that the -barrel of the

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homologous protein in Neurospora crassa contains only a single cysteine 33. Carrier proteins traverse the Tom40 channel in a loop structure 34,41,59 and if both polypeptides in transit adopt

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an -helical conformation, each -helix would require a space of about 12 Å diameter. It is therefore remarkable that a reaction of the cysteine residues with N-Ethylmaleimide (NEM)

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did not cause a complete block of the import channel. In fact, the import efficiency of VMC1 and of other carrier proteins was reduced by only 20 to 30%. Considering that NEM

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molecules have a diameter of about 4 Å, the efficient import reactions demonstrate that the two polypeptide chains of the carrier loop structures do not translocate through a tightly fitting tube. The import channel may have some flexibility, alternatively, and more likely 68,

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the polypeptide chains – including the potentially -helical transmembrane segments of the

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carrier proteins - may adopt a more extended and thus less space-filling conformation and pass the narrow pore more easily.

The impact of mimivirus as a human pathogen is still unclear 2,69. Mimivirus DNA was isolated from several patients with pneumonia, and it was proposed that mimivirus-infected amoeba may have a substantial clinical significance, at least in patients at risk of opportunistic infections 2,70,71,72,73. However, other studies suggested that the relevance of mimivirus in diseases of the respiratory tract is rather limited 74,75,76,77. On the other hand, a recent 13

ACCEPTED MANUSCRIPT investigation on viruses in tissue samples of cancers of immunosuppressed patients revealed that a multitude of viruses was detectable in the tumor specimens, surprisingly with sequences related to Mimiviridae being the most prevalent 78. In this context it may be of interest that mimivirus-encoded VMC1 shows efficient targeting to mitochondria also in (human) HeLa cells (Fig. 1). In direct comparison, VMC1 is imported into mitochondria with similar (Fig. 2)

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or even with higher efficiencies (Fig. 5) than endogenous mitochondrial carrier proteins.

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VMC1 should thus be able to contribute to mimivirus replication also in human tissues.

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Materials and Methods

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Expression of VMC1 in HeLa cells. HeLa cells were cultured in DMEM (Sigma Aldrich) with 10% fetal calf serum, 1% L-Glutamine and 1% Penicillin-Streptomycin (Sigma Aldrich)

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at 37°C in an incubator with an atmosphere of 8% CO2. For Doxycycline-inducible

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expression of His tagged proteins, the corresponding DNA fragments were cloned into the vector pTET zeo+ one. The cells were transfected using X-treme GENE HP DNA transfection

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reagent (Roche) and cultured for about three months to get a stable population of transfected cells in a medium containing Zeocin (InvivoGen).

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Immunofluorescence labeling was performed following a conventional protocol. Briefly, 24 (48,72) hours after induction with Doxycycline, cells grown on glass coverslips were rinsed with PBS and fixed for 20 min with a 3% paraformaldehyde solution followed by a 5 min permeabilization in PBS + 1% Triton X-100. The coverslips were 30 min incubated in a 1:500 dilution of mouse anti-His antibody and a rabbit anti-Trap1 antibody (1:350) Afterwards the cells were rinsed with PBS. Then the secondary antibodies were used: a green fluorescence goat anti mouse antibody (1:300) and the red fluorescence goat anti rabbit antibody (1:200). The coverslips were finally rinsed and mounted in Mowiol medium combined with DAPI 14

ACCEPTED MANUSCRIPT (4′,6-diamidino-2-phenylindole) prepared according to the manufacturer`s instructions. Pictures were acquired on a Axioplan 2 imaging microscope (Zeiss) with an alpha Plan Fluar 100x (63x)/1.45 objective under oil immersion. The relative amounts of reactive oxygen species (ROS) were determined using the ROSsensitive fluorescent reagent 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) 35. HeLa

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cells were grown in 96 well plates for 24 h in the presence of 100 µM DCFH-DA, protein

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expression was subsequently induced by 100 ng/ml doxycycline for 96 h. ROS-dependent

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fluorescence was determined using a plate reader. The number of vital cells was determined using the dye Neutral Red (3-Amino-7-dimethylamino-2-methylphenazine hydrochloride) for

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labelling.

Expression of VMC1 in yeast. Yeast cells were grown in YPG medium (1% (w/v) yeast

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extract, 2% (w/v) bacto-peptone, pH 5.0, containing 3% (v/v) glycerol). The DNA fragment encoding VMC1 was inserted into the vector pUG36 (Dr. Hegemann, University of

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Düsseldorf, Germany) for constitutive expression of a hybrid protein containing an enhanced

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green fluorescent protein yEGFP3 79 from a MET25 promotor, or into the vector pYES2 for galactose-induced expression.

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Synthesis of radiolabelled VMC1 in reticulocyte lysate and import into isolated mitochondria. Isolation of yeast mitochondria and procedures of mitochondrial protein

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import are described by Rassow 80 and Papatheodorou et al. 39. The preparation of rat liver mitochondria was carried out as described by Domańska et al. 81. For protein import, 35Slabelled proteins were synthesized in rabbit reticulocyte lysates (Promega) in the presence of S-methionine (Hartmann Analytik). Isolated mitochondria of rat liver or yeast (30 g of

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protein) were incubated with the reticulocyte lysates (2-4 l) in BSA-buffer (3% [w/v] BSA, 250 mM sucrose, 80 mM KCl, 5 mM MgCl2, 10 mM MOPS/KOH, 2 mM NADH, pH 7.2) in a final volume of 50 l at 25°C. In some experiments, 2 M valinomycin was added to

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ACCEPTED MANUSCRIPT dissipate the mitochondrial membrane potential. For protease treatment, the samples were cooled to 0°C and incubated with 75 g/ml proteinase K for 10 min at 0°C. Proteolysis was stopped by addition of 2 mM PMSF (Phenylmethylsulfonylfluoride) for 5 min at 0°C. Mitochondria were reisolated by centrifugation for 10 min at 16.000 g and analysed by SDSPAGE or by BN-PAGE.

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BN-PAGE (Blue Nativegelelectrophoresis). The samples used for BN-PAGE contained 50

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g mitochondrial protein, 1% digitonin, 10% glycerol (v/v), 50 mM NaCl, 0.1 mM EDTA, 20

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mM Tris-HCl pH 7.4 in a volume of 40 l. The samples were centrifuged 15 min at 16.000 g, the supernatants were mixed with 4 l 500 mM -aminocaproic acid, 5% (w/v) Coomassie

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Blue G-250 (Serva), 100 mM Bis-Tris pH 7.0 and loaded on a 4 - 16% acrylamide gradient gel (SERVAGel Kat. Nr. 43252). The gel chamber was cooled in a water bath at 0°C.

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Binding of mitochondrial preproteins to isolated receptor proteins was studied following the protocol of Brix et al. 42.

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Sucrose density gradient centrifugation. Mitochondrial membrane vesicles were obtained

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using a Sonifyer (Branson), subsequent separation by a sucrose density step gradient followed the procedure described by Domańska et al. 81.

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N-Ethylmaleimide (NEM, Sigma) was freshly dissolved in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH pH 7.2 (SEM buffer) and incubated with mitochondria (30 g protein in

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a final volume of 50 l SEM buffer) for 20 min at 25°C. The reaction was stopped by reisolation of the mitochondria by centrifugation. The hybrid protein b2(1-47)DHFR contains an N-terminal segment of yeast cytochrome b2 (residues 1-47) fused to the dihydrofolate reductase (DHFR) of the mouse 68. The protein was used for competition experiments as described by Motz et al. 54.

16

ACCEPTED MANUSCRIPT Acknowledgements. We thank Edmund Kunji, University of Cambridge, for kindly sharing a VMC1 plasmid, and we thank Ralf Erdmann, Ruhr-University Bochum, Peter Rehling, University of Göttingen, and Nikolaus Pfanner, Nils Wiedemann and Jan Brix, University of Freiburg, for kindly sharing yeast strains and antisera. This work was supported by funds from MIUR (Ministero

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dell'Istruzione dell'Università e della Ricerca) (V.Z. and A.F.), the DFG (grant RA 702/4-1)

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(J.R.), and a FoRUM grant of the Medical Faculty of the Ruhr-University Bochum (P.G).

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ACCEPTED MANUSCRIPT Figure legends

Fig. 1. Expression of VMC1 in HeLa cells. (a) Intracellular distribution of VMC1. Expression was induced by addition of doxycycline for 48 h. The cells were subsequently fixed and VMC1 was labelled using an antibody directed against the C-terminal (His)10-tag of the

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protein and a green fluorescent secondary antibody. The mitochondria were labelled using an

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antibody against Trap1 (a mitochondria-specific Hsp90 chaperone) and a red fluorescent secondary antibody (upper panel) or the fluorescent dye Mitotracker Orange (lower panel).

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The nuclear DNA was visualized by DAPI (blue colour). Bar, 10 m. (b) Assay for

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intracellular reactive oxygen species (ROS). HeLa cells were grown in the presence of 100 µM DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate) and treated with CCCP (carbonyl

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cyanide m-chlorophenylhydrazone) and Antimycin A at the concentrations as indicated for 48 h. ROS-dependent fluorescence was determined using a plate reader. Mean cell numbers were

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determined by neutral red viability testing. Shown is the calculated ratio of fluorescence per

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cell number in individual samples in relation to average fluorescence per cell number in the absence of CCCP and Antimycin A. The median was calculated from 11 samples. (c)

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Determination of ROS following expression of VMC1. HeLa cells were stably transfected with plasmids encoding VMC1, the mitochondrial outer membrane protein Tom20, or with

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the empty vector, respectively. Both proteins contained a C-terminal (His)10-tag. Expression was induced by addition of 100 ng/ml doxycycline. ROS levels of doxycycline-treated cells were determined after 96 h as in (b). The values were calculated in relation to parallel noninduced control samples (no doxycycline). The median was calculated from the results of 8 biological replicates (independent experiments). (d). Test of protein expression. HeLa cells were grown as in (c) and subsequently isolated for SDS-PAGE and western blotting. The membranes were decorated with antibodies directed against the (His)10-tag of the proteins.

30

ACCEPTED MANUSCRIPT Fig. 2. Import of radio-labelled VMC1 into isolated rat liver mitochondria. (a) Degradation of newly synthesized VMC1 by proteinase K. 35S-labelled VMC1 was synthesized in reticulocyte lysate. Samples were incubated with proteinase K (PK) at the concentrations as indicated for 10 min at 0°C. (b) Import into mitochondria. VMC1 and N. crassa AAC (ADP/ATP carrier) were synthesized in reticulocyte lysate and incubated with isolated rat

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liver mitochondria at 25°C as indicated. The mitochondria were subsequently incubated with

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75 g/ml proteinase K, proteolysis was stopped by addition of PMSF, and the mitochondria

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were reisolated by centrifugation for subsequent SDS-PAGE. The relative amounts of radiolabelled protein were determined by digital autoradiography using a phosphorimager,

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standard deviations were calculated from the results of three independent experiments.

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Fig. 3. Expression of VMC1 in the yeast S. cerevisiae. (a) VMC1 fused to enhanced green fluorescent protein (yEGFP, 79) was expressed in yeast cells. For fluorescence microscopy, the

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mitochondria were labelled by addition of the dye Mitotracker Orange. Bar, 5 m. (b)

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Expression of VMC1 (lanes 1 and 2) and N. crassa AAC (lanes 3 and 4) in yeast, both proteins carrying an amino-terminal (His)10-tag for detection by a specific antibody.

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Expression was induced by galactose for 1 h, the cells were lysed and the proteins precipitated by trichloroacetic acid (TCA) for analysis by SDS-PAGE and immune blotting. (c) Growth of

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yeast cells on agar plates in presence of glucose or galactose, respectively. Cell suspensions were diluted as indicated.

Fig. 4. Import of radiolabelled AAC and VMC1 into isolated yeast mitochondria. (a) Protease-protection assay. Mitochondria were incubated with reticulocyte lysates containing 35

S-labelled VMC1 or AAC at 25°C for the times as indicated and subsequently treated with

75 g/ml proteinase K. The mitochondria were reisolated and proteins were separated by

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ACCEPTED MANUSCRIPT SDS-PAGE, including a defined sample of reticulocyte lysate as a reference for quantification. Relative amounts of radiolabelled AAC were determined using a phosphorimager, SD, n = 3. (b) Assay for binding to mitochondria. Reticulocyte lysates containing 35S-labelled VMC1 or AAC and isolated mitochondria were preincubated separately with apyrase to degrade the ATP of the samples. The reticulocyte lysates were then

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incubated with the mitochondria for different times at 25°C and the mitochondria were

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reisolated for SDS-PAGE and digital autoradiography. Samples lacking mitochondria were

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processed in parallel under the same conditions to determine the amounts of aggregated proteins (< 1% of VMC1 and < 3% of the total amounts of AAC). In calculating the ratios of

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bound protein, the values of aggregation were subtracted, SD, n = 3. (c) Binding of radiolabelled proteins to purified cytosolic domains of mitochondrial import receptors Tom70,

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Tom20 or Tom22. Receptor proteins bound to a Ni2+-NTA resin were incubated with reticulocyte lysate containing 35S-labelled AAC, VMC1 or Su9-DHFR (amino-terminal

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residues 1-69 of N. crassa ATP synthase subunit 9 fused to mouse dihydrofolate reductase)

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for 40 min at 30°C. The Ni2+-NTA beads were rinsed with buffer, and associated proteins were subsequently eluted by 1 M imidazole, precipitated by trichloroacetic acid (TCA) and

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separated by SDS-PAGE. A phosphorimager was used for quantification. The total amounts of 35S-labelled protein per sample were set to 100%, n = 3. (d) Sucrose density centrifugation

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of membrane vesicles. VMC1 carrying a (His)10-tag was expressed in yeast and the mitochondria were isolated. The membranes were disrupted by sonication, and the membrane vesicles were separated by sucrose density centrifugation. Following SDS-PAGE and transfer to nitrocellulose, specific antisera were used to label VMC1 and the marker proteins Tom40 (outer membrane) and Tim23 (inner membrane). (e) Blue native gelelectrophoresis (BNPAGE). 35S-labelled VMC1 was imported into isolated yeast mitochondria in the presence or absence of ATP as indicated. Valinomycin was added to samples 1, 2, 4 and 5 to dissipate the mitochondrial membrane potential (- ), proteinase K was added to samples 4-6 to degrade 32

ACCEPTED MANUSCRIPT VMC1 outside the mitochondrial membranes (+ PK). The mitochondria were lysed in the presence of 1% digitonin and the protein complexes were separated by BN-PAGE. Radiolabelled VMC1 was detected by digital autoradiography. Stage II, receptor-bound; stage III, location in the intermembrane space; stage V, mature protein in the inner membrane, 40,41,46

. (f) Inhibition of protein import by blocking of the outer membrane import channel.

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Isolated yeast mitochondria were preincubated with b2(1-47)-DHFR (a fusion protein

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comprising residues 1-47 of yeast cytochrome b2 fused to mouse dihydrofolate reductase, 54).

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As indicated, up to 1 g of b2(1-47)-DHFR were used in sample volumes of 50 l. The mitochondria were subsequently incubated for 10 min at 25°C with reticulocyte lysate

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containing 35S-labelled mimivirus VMC1, N. crassa AAC, N. crassa F1 (subunit  of the mitochondrial ATP synthase) or S. cerevisiae Ugo1 (a mitochondrial outer membrane

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protein), respectively. The mitochondria were reisolated, incubated with proteinase K, and analysed by SDS-PAGE and autoradiography. The amounts of radiolabelled protein imported

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in the absence of b2(1-47)-DHFR were set to 100% (control).

Fig. 5. Inhibition of protein import by pretreatment of mitochondria with NEM (N-

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ethylmaleimide). (a) Cross section of yeast mitochondrial outer membrane protein Tom40. Indicated are the membrane-spanning -strands of the protein (1 – 19), the localization of

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cysteine residues within the -barrel (red), and residues of close proximity to translocating polypeptides (black; labelled according to data published by Shiota et al. 32). (b) Distribution of cysteine residues within membrane-spanning Tom40 -strands. (c) Inhibition of protein import by NEM. Isolated yeast mitochondria were pretreated with trypsin at 0°C to degrade outer membrane receptor proteins, reisolated, and incubated with increasing concentrations of NEM for 20 min at 25°C as indicated. The mitochondria were again reisolated, valinomycin was added to dissipate the membrane potential, and 35S-labeled AAC was imported for 10 min 33

ACCEPTED MANUSCRIPT at 25°C. (d) Effects of NEM pretreatment on import of AAC, VMC1 and DIC. Mitochondria were pretreated with trypsin and with 2 mM NEM, reisolated, and incubated with 35S-labeled N. crassa AAC, mimivirus VMC1 or S. cerevisiae dicarboxylate carrier (DIC) in presence of valinomycin for 10 min at 25°C. The mitochondria were treated with proteinase K, and reisolated for subsequent analysis by SDS-PAGE and digital autoradiography. The average

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amounts of radiolabelled protein imported in the absence of NEM were set to 100% (control),

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SD, n = 3.

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ACCEPTED MANUSCRIPT

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Highlights • Data are presented on VMC1, a nucleotide carrier encoded by gene L276 of Mimivirus, a giant DNA virus • VMC1 is shown to target the mitochondrial inner membrane • Translocation into the mitochondria is mediated by the mitochondrial outer membrane protein Tom40 • Import of VMC1 into the mitochondria continues after partial occlusion of the import channel • VMC1 is suggested to pass through the narrow import channel in an extended conformation

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5