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The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria
J. Mol. Biol. (2012) 423, 590–599
http://dx.doi.org/10.1016/j.jmb.2012.07.018 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria Markus Hildenbeutel 1 , Melanie Theis 1 , Melanie Geier 2 , Ilka Haferkamp 2 , H. Ekkehard Neuhaus 2 , Johannes M. Herrmann 1 ⁎ and Martin Ott 3 1
Division of Cell Biology, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany Division of Plant Physiology, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany 3 Research Group Membrane Biogenesis, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany 2
Received 4 June 2012; received in revised form 18 July 2012; accepted 20 July 2012 Available online 27 July 2012 Edited by J. Bowie Keywords: carrier proteins; membrane biogenesis; mitochondria; Oxa1 protein import
Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperaturesensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin. © 2012 Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: [email protected]. Present addresses: M. Hildenbeutel, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, SE-106 91 Stockholm, Sweden; M. Ott, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, SE-106 91 Stockholm, Sweden. Abbreviations used: DTT, dithiothreitol; TIM, translocase of the inner membrane; TOM, translocase of the outer membrane.
Introduction Proteins of the inner membrane of mitochondria represent a mosaic of dual genetic origin: The mitochondrial genome encodes a small number of hydrophobic membrane proteins that are integrated into the inner membrane in a co-translational reaction. 1,2 The vast majority of mitochondrial proteins is nuclear encoded and synthesized on cytosolic ribosomes. 3–5
0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.
Oxa1 Facilitates Carrier Biogenesis
Membrane insertion of mitochondrial translation products is facilitated by the inner membrane protein Oxa1. 6–8 Oxa1 is an integral membrane protein with five transmembrane helices and a Cterminal ribosome-binding domain. 1,2 It belongs to a family of proteins with representatives in bacteria (YidC proteins), mitochondria (Oxa1 and Cox18) and plastids (Alb3 and Alb4). Deletion mutants of Oxa1 and YidC show a block in membrane integration of several proteins, suggesting that Oxa1/YidC/Alb3 proteins function as insertases that facilitate membrane integration of transmembrane segments. 6,8–10 In vitro reconstitution experiments of recombinant YidC confirmed its insertase activity. 11,12 In addition to its role as insertase, YidC was proposed to be critical for the folding of some membrane proteins that are initially inserted in an YidC-independent manner. 13,14 A more general role of YidC in the quality control of membrane proteins would be consistent with the severe stress response and upregulation of chaperones and proteases in YidC-depleted cells that were recently observed in proteome-wide analyses. 15–17 However, direct evidence for an insertion-independent folding function of YidC or Oxa1 is lacking since insertion and folding of membrane proteins are difficult to distinguish experimentally. In addition to its role in co-translational protein insertion, Oxa1 was shown to facilitate membrane integration of some nuclear‐encoded proteins that embark on a so-called conservative sorting pathway. 18–20 These substrates are initially imported into the matrix by the TIM23 (TIM, translocase of the inner membrane of mitochondria) complex, the general transporter for matrix proteins. After full or partial translocation, they insert into the inner membrane in an export-like process. All nuclear‐ encoded Oxa1 substrates identified so far (Oxa1, Cox18, Su9/Foc and Mdl1) have homologs in bacteria and presumably evolved from components that were synthesized in the cytosol of the endosymbiotic progenitors of mitochondria. In contrast to conservatively sorted proteins, members of the mitochondrial carrier family use a completely different import pathway that deviates from the import route of matrix proteins after translocation across the outer membrane. They insert from the intermembrane space into the inner membrane in a process relying on a specific inner membrane translocase, the TIM22 complex. 3,21,22 The carrier family contains about 35 members in yeast and more than 50 in humans. 23,24 Carrier proteins are characterized by six transmembrane domains that are arranged in three repetitive modules that tightly interact to form a channel-like structure. 25 The mitochondria-specific lipid cardiolipin plays an important role in the biogenesis, stability and function of carrier proteins. 26–28 Carrier proteins are a signature family of eukaryotic proteins
591 and developed in the cause of the endosymbiont-toorganelle transition of mitochondrial progenitors to connect metabolic and nucleotide networks within the eukaryotic cell. 29–32 Here, we report that Oxa1 plays an unexpected role in the biogenesis of mitochondrial carrier proteins. Upon depletion of Oxa1, carrier proteins are imported into mitochondria with reduced efficiency and accumulate to significantly reduced steady‐state levels. These observations suggest that Oxa1 plays a more general role in inner membrane biogenesis than previously proposed.
Results and Discussion Deletion of Oxa1 leads to reduced levels of the ADP/ATP carrier Oxa1 is known to mediate the insertion of mitochondrially encoded and conservatively sorted inner membrane proteins. 8,18,33,34 Hence, Oxa1 deletion mutants (Δoxa1) show reduced levels of components of respiratory chain complexes (Fig. 1a, Cox2, Cor2, Rip1 and Shy1). Moreover, proteins with conservatively sorted domains rely on Oxa1 and are reduced in the Δoxa1 mitochondria (Fig. 1a, Atm1). 18 In contrast, proteins of the outer membrane, the intermembrane space and the matrix and most proteins of the inner membrane are not affected in Δoxa1 strains (Fig. 1a, Tom70, Mrpl40, Phb2, Yme1, TIM23 and TIM22). Surprisingly, we repeatedly observed significantly reduced levels of ATP/ADP carrier (Fig. 1a, Aac2) in Δoxa1 strains, which was unexpected since carrier proteins reach the inner membrane after insertion from the intermembrane space by the TIM22 translocase. A relevance of Oxa1 for TIM22-mediated protein biogenesis was not reported so far. To further analyze the relevance of Oxa1 for biogenesis of the ATP/ADP carrier, we directly measured the efficiency of ADP transport into wild type and Δoxa1 mitochondria (Fig. 1b). To this end, we incubated de-energized mitochondria from wild type and Δoxa1 cells with 32P-labeled ADP and measured uptake of the radioactive nucleotide. Compared to wild type, ADP uptake was considerably reduced in Δoxa1 mitochondria. Hence, the absence of Oxa1 prevents the generation of normal ATP/ADP carrier levels in mitochondria and, consequently, efficient nucleotide transport. It should be noted that the absence of respiratory chain complexes in Δoxa1 mitochondria leads to a severely reduced membrane potential in this mutant (Fig. 1c). Thus, it remains possible that the pronounced effect of the Oxa1 deletion on carrier accumulation is an indirect effect due to the decreased mitochondrial membrane potential or due to an altered lipid composition in Δoxa1
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Fig. 1. Aac2 levels are reduced in Δoxa1 mitochondria. (a) Mitochondria were isolated from W303 wild type (wt) and an Oxa1 deletion strain (Δoxa1). Protein levels were analyzed by Western blotting using the indicated antibodies. Diminished steady‐state levels of proteins are depicted by arrowheads. IM, inner membrane; OM, outer membrane. (b) Δoxa1 mitochondria show considerably reduced uptake of radioactively labeled [α 32P]ADP. Values for carrierindependent ADP uptake in the presence of atractyloside and bongkrekic acid were subtracted. Shown are mean values of three independent experiments. (c) Δoxa1 mitochondria show a strongly reduced membrane potential. Freshly isolated mitochondria were energized by addition of 2 mM NADH before the membrane potential was analyzed using the dye JC-1. The membrane potential was dissipated again by addition of 25 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Shown are mean values of three measurements. (d and e) The Aac2 levels in Δoxa1 mitochondria are lower than that in mitochondria of a Δcrd1 mutant or of other respiration-deficient strains. Equal amounts of isolated mitochondria of the indicated strains were loaded on SDS gels. For (e), levels of Aac2 were quantified by Western blotting.
mitochondria. 35,36 However, the levels of Aac2 in Δoxa1 mitochondria are lower than those in mitochondria lacking the cardiolipin synthase Crd1 (Fig. 1d) or in mitochondria of other respiration‐ deficient strains (Fig. 1e). Surprisingly, even rho 0 strains in which mitochondrial genomes and, hence, functional enzymes of the respiratory chain are completely absent contain higher levels of Aac2 than Δoxa1 mitochondria.
A temperature-sensitive allele allows the specific depletion of Oxa1 The low membrane potential of Δoxa1 mitochondria makes it difficult to exclude indirect effects on the biogenesis of carrier proteins, which relies on the energization of the inner membrane. 37 In the past, the use of temperature-sensitive Oxa1 variants proved to be a powerful strategy to circumvent this
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problem. 18,38–40 In the oxa1 alleles used so far, incubation at 37 °C inactivated Oxa1 at least partially allowing to analyze its relevance in the context of an otherwise unaltered organelle. Unfortunately, a residual activity of the heat-treated Oxa1 protein cannot be excluded in these strains. To this end, we searched for novel temperature-sensitive oxa1 mu-
tants. We identified an Oxa1 point mutant in which a conserved histidine residue in position 121 was replaced by a leucine residue (Fig. 2a). This mutation provoked a clear-cut temperature-sensitive growth phenotype on non-fermentable carbon sources (Fig. 2b). This respiration deficiency was due to a selective loss of Oxa1 upon incubation at 37 °C,
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Fig. 2. An H121L mutant of Oxa1 leads to a temperature-sensitive phenotype. (a) A section of an alignment of Oxa1 sequences is presented to show the conservation of the histidine 121 residue. Sequences were used of S. cerevisiae, Kluyveromyces lactis, Schizosaccharomyces pombe, Mus musculus and Homo sapiens. Invariant residues are highlighted. (b) Wild type and Oxa1ts cells were grown to log phase. We dropped 10‐fold serial dilutions on fermentative (glucose) or non-fermentative (glycerol) medium. The plates were incubated at 25 or 37 °C as indicated. (c) Incubation of Oxa1ts cells leads to the depletion of Oxa1. Cells were grown to log phase at 25 °C, washed and further incubated at either 25 or 37 °C for the times indicated. The levels of Oxa1 and Tom70 in whole cell extracts were analyzed by Western blotting. (d) Mitochondria were incubated from wild type or Oxa1ts mutants grown at 25 °C. Following incubation at 25 or 37 °C for the times indicated, we assessed the levels of Oxa1 and Mrp20 by Western blotting. (e) Depletion of Oxa1 prevents maturation of newly synthesized Cox2 precursor (pre-Cox2). Translation products were labeled with [ 35S]methionine in isolated mitochondria 41 for the indicated time periods, separated by SDS-PAGE and detected by autoradiography. Cyt b, cytochrome b.
Oxa1 Facilitates Carrier Biogenesis
594 which was observed when either cells or isolated mitochondria of the oxa1ts strain were incubated at 37 °C (Fig. 2c and d). To analyze the activity of Oxa1 in the Oxa1ts mutant more directly, we grew yeast cells of the wild type or the Oxa1ts mutant at 25 °C and shifted the cultures for 3 h to 25 or 37 °C prior to the preparation of mitochondria. Then, we followed the processing of the mitochondrially encoded protein Cox2 after synthesis in isolated mitochondria (Fig. 2e). Following its insertion, the leader peptide of Cox2 precursor (pre-Cox2) is cleaved by Imp1/Imp2 protease in the intermembrane space. 42 In the absence of Oxa1, membrane insertion of pre-Cox2 is strongly reduced. 8,33 Pre-Cox2 was efficiently matured in Oxa1 and Oxa1ts mitochondria at permissive conditions (Fig. 2e). In contrast, nonprocessed pre-Cox2 accumulated in Oxa1-depleted mitochondria, indicating the absence of Oxa1 activity in these mitochondria. Hence, the Oxa1ts mutant identified in this study allows switching off Oxa1 activity by heat treatment. Depletion of Oxa1 in the Oxa1ts mutant does not influence the membrane potential Next, we directly assessed the protein levels in mitochondria isolated from wild type or Oxa1ts cells after preincubation for 3 h at permissive or restrictive conditions (Fig. 3a). Noteworthy, no Oxa1 could be detected in mitochondria isolated from heatexposed Oxa1ts cells, indicating the efficient deple-
(a)
tion of the Oxa1 mutant protein at 37 °C. Misfolded Oxa1 was shown to be unstable and to be rapidly degraded by mitochondrial proteases. 40,43 Importantly, at these early stages of Oxa1 depletion, respiratory chain components and other mitochondrial proteins were still present at unaltered levels (Fig. 3a). Moreover, this heat treatment did not considerably reduce the membrane potential of these mitochondria, indicating that the inner membrane is still properly energized (Fig. 3b). We next tested the activity of Oxa1-depleted mitochondria in the uptake of 32P-labeled ADP. Similar amounts of ADP were taken up independent of the presence or absence of Oxa1 (Fig. 3c), showing that the chosen regime to deplete Oxa1 does not impair ATP/ADP carrier activity. Taken together, these data show that mitochondria isolated from this Oxa1ts strain at 37 °C specifically lack Oxa1 but contain a functional respiratory chain and an intact inner membrane. Efficient import of carrier proteins depends on the presence of Oxa1 Mitochondria of the heat-treated Oxa1ts mutant allowed us to investigate whether the biogenesis of carrier proteins is affected by the absence of Oxa1. To this end, we followed the import of in vitro synthesized, radioactively labeled precursor proteins into isolated mitochondria (Fig. 4). As expected, matrix proteins such as the ATPase subunit F1β and
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Fig. 3. Heat exposure of Oxa1ts cells leads to rapid depletion of Oxa1 but does not impair the energization of the inner membrane. (a) Degradation of Oxa1 in the Oxa1ts strain has no effect on the levels of other inner membrane proteins. Yeast cells were grown at 25 °C. Prior to isolation of mitochondria, yeast cells were either kept at 25 °C or shifted to 37 °C for 3 h. Mitochondria were analyzed by Western blotting using the antibodies indicated. (b) The membrane potential in the Oxa1ts mitochondria was analyzed as in Fig. 1c. (c) ADP uptake efficiency was measured as described in Fig. 1b in Oxa1ts mitochondria that were pretreated or not pretreated at 37 °C to induce Oxa1 degradation.
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Su9 1-69-DHFR were efficiently imported, irrespective of the presence or absence of Oxa1 (Fig. 4a). These results confirmed the intactness of the mitochondrial inner membrane because import of matrix
Fig. 4. Efficient import of members of the mitochondrial carrier family depends on Oxa1. Mitochondria of the indicated strains were incubated for different time periods with different radiolabeled preproteins. Samples were treated with proteinase K and analyzed by autoradiography. The signals were quantified and corrected for the import rates of the matrix protein F1β. Preproteins used were the matrix proteins F1β and Su9 1-69DHFR (a), Aac2 of S. cerevisiae (b), Aac2 of N. crassa (c), the phosphate carrier Pic2 (d) and the dicarboxylate carrier Dic1 of S. cerevisiae (e).
proteins via the TIM23 pathway relies on a membrane potential. Next, we tested whether import of Aac2 into these mitochondria occurs normally. Aac2 was efficiently
Oxa1 Facilitates Carrier Biogenesis
596 imported into mitochondria containing Oxa1 but only with reduced efficiency into mitochondria lacking Oxa1 (Fig. 4b). This dependency on Oxa1 for import was not restricted to yeast Aac2. When Aac2 from the filamentous fungus Neurospora crassa was imported into yeast mitochondria, efficiency of import was decreased as well (Fig. 4c). Moreover, also the import of other members of the mitochondrial carrier family such as the phosphate carrier Pic2 and the dicarboxylate carrier Dic1 was strongly reduced in the absence of Oxa1 (Fig. 4d and e). To exclude indirect effects of slightly reduced levels of the mitochondrial membrane potential, we
performed co-import experiments of a member of the mitochondrial carrier family and F1β into isolated mitochondria in the same reaction. The import of F 1 β requires a robust membrane potential 44 making F1β a very good control for the energy state of the inner membrane. As shown in Fig. 5a and b, the amounts of imported F1β were comparable in the absence or presence of Oxa1, whereas Aac2 from Saccharomyces cerevisiae and N. crassa as well as Pic2 and Dic1 required Oxa1 for efficient import. We therefore concluded that efficient mitochondrial biogenesis of carrier proteins depends on Oxa1. Nevertheless, even in the absence
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Fig. 5. Oxa1 depletion specifically impairs the biogenesis of carrier proteins. (a) Mitochondrial carrier proteins and the nuclear‐encoded ATPase subunit F1β were simultaneously incubated with isolated mitochondria for 15 min. Non-imported protein was degraded by proteinase K treatment. Samples were analyzed by autoradiography. (b) The experiment shown in (a) was repeated three times and the signals were quantified. The values indicate the ratios of carrier import relative to the import efficiencies observed for F1β. (c) Aac2 reaches a membrane-embedded location after import into Oxa1ts mitochondria. Aac2 was imported for 30 min into mitochondria of mock- or heat-treated cells. We used a relatively long import time here in order to accumulate significant amounts of Aac2 also in Oxa1-depleted mitochondria. Mitochondria were reisolated and either directly loaded onto the SDS gel (T, total) or separated into a soluble (S) and membrane pellet (P) fraction by carbonate treatment. The samples were analyzed by autoradiography (Aac2) and Western blotting using antibodies against the membrane protein Cox2 and the soluble protein Mprl40. (d) Model for carrier biogenesis. At this stage, it remains unclear whether Oxa1 plays a direct (left) or indirect (right) role during carrier biogenesis.
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of Oxa1, Aac2 finally reached the inner membrane and, after prolonged import periods, could be identified in a carbonate-resistant fraction together with integral membrane proteins such as Cox2 (Fig. 5c).
yeast cultures were either kept at 25 °C or shifted to 37 °C for 3 h in the presence of 20 μM cycloheximide. Mitochondria were isolated as previously described. 46
Conclusion
Radioactively labeled [α 32 P]ADP was synthesized e n z y m a t i c a l l y u s i n g h e xok i n a s e a s d e s c rib ed previously. 47 For ADP uptake measurements, freshly isolated mitochondria were added to 100 μl of transport buffer [20 mM Hepes–KOH (pH 7.0), 15 mM KPi (pH 7.0), 80 mM KCl and 0.6 M sorbitol] containing [α 32P]ADP (Hartmann analytics). Reactions were incubated for 5 s before mitochondria were collected on a filter membrane and extensively washed with ice-cold transport buffer. Radioactivity in each sample was quantified by scintillation counting. To determine background levels, we determined ADP uptake in mitochondria that were pretreated with atractyloside and bongkrekic acid for 5 min to block ATP/ADP carrier activity. Background values were subtracted.
Here, we show that Oxa1 plays a critical role in the biogenesis of mitochondrial carrier proteins. At this stage, it is not clear whether Oxa1 physically interacts with carrier proteins during their import or folding in the inner membrane, and it remains possible that Oxa1 influences the import of carrier proteins only indirectly, for example, by changes in the lipid composition of the mitochondrial membranes (Fig. 5d). However, we regard this as unlikely since the temperature-sensitive mutant that was used in this study, shows wild‐type levels of respiratory chain complexes, of the membrane potential and of the import of matrix proteins even at restrictive conditions. The data shown here indicate that the presence of Oxa1 in the inner membrane is a direct prerequisite for carrier import. Hence, Oxa1 does not only promote the export of proteins from the matrix into the inner membrane but also exhibits a function in the biogenesis of membrane proteins that integrate into the inner membrane via the TIM22 complex. Although we do not know the precise biochemical role of Oxa1 in this process, it appears likely that Oxa1 promotes the folding of newly imported carrier proteins in the inner membrane. A role in the folding of membrane proteins was also proposed for YidC, which was shown to facilitate the biogenesis of proteins that are initially inserted into the bacterial membrane in a YidC-independent process. 13,14,16,17 In summary, we conclude that the substrate spectrum of Oxa1 is presumably much broader than initially expected and not limited to proteins of bacterial origin.
Materials and Methods Yeast strains and growth media All strains used in this study were isogenic to the wild‐ type strain W303. OXA1 was deleted with a HIS3 selection cassette. Oxa1ts strains were derived from a Δoxa1 strain transformed with the plasmid pRS314 45 carrying the endogenous Oxa1 promoter and the temperature-sensitive Oxa1 allele with a H121L mutation. All yeast cultures were grown at 25 °C in lactate medium [0.3% yeast extract, 0.1% KH2PO4, 0.1% NH4Cl, 0.05% CaCl2⋅H2O, 0.05% NaCl, 0.06% MgSO4⋅H2O, 0.0003% FeCl3 and 2% lactate (pH 5.5) (adjust with KOH)] or YP medium (1% yeast extract and 2% peptone) supplemented with either 2% glucose, galactose or glycerol. For the isolation of mitochondria,
Uptake of radioactively labeled ADP into isolated mitochondria
Membrane potential measurements For membrane potential measurements, freshly isolated mitochondria were diluted to an OD595 of 0.25 in a total of 400 μl transport buffer. Mitochondria were energized by addition of 2 mM NADH and incubation of the reaction at 30 °C for 30 s. Upon addition of JC-1 (Calbiochem) to a final concentration of 0.5 μg/ml, the increase in fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 590 nm for 330 s. To dissipate the membrane potential, we added 25 μM carbonyl cyanide m-chlorophenyl hydrazone, and we monitored the decrease in fluorescence for additional 90 s. Import of in vitro synthesized preproteins into isolated mitochondria Proteins were synthesized in reticulocyte lysate as previously described. 48 Per reaction, 40 μg of mitochondria were diluted in 40 μl of import buffer [50 mM Hepes– KOH (pH 7.4), 0.6 M sorbitol, 80 mM KCl, 10 mM Mg acetate, 2 mM KPi and 1 mM MnCl2] in the presence of 2 mM ATP and 2 mM NADH. Import reactions were started by addition of 2 μl of preprotein-containing reticulocyte lysate. Import reactions were stopped by 10‐ fold dilution in ice‐cold SH (sorbitol–Hepes) buffer. Nonimported proteins were removed by addition of proteinase K (100 μg/ml) and incubation for 30 min on ice. Protease treatment was stopped by addition of 1.5 mM PMSF. Mitochondria were isolated by centrifugation and washed three times with SH–KCl buffer [20 mM Hepes– KOH (pH 7.4), 0.6 M sorbitol and 80 mM KCl].
Acknowledgements We thank Jan Riemer for critical reading of the manuscript. We are grateful to Sabine Knaus and
Oxa1 Facilitates Carrier Biogenesis
598 Andrea Trinkaus for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 530 and FOR 967), the Landesstiftung für Innovation Rheinland-Pfalz and the Landesforschungsschwerpunkt Membrantransport.
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29. Dolezal, P., Aili, M., Tong, J., Jiang, J. H., Marobbio, C. M., Lee, S. F. et al. (2012). Legionella pneumophila secretes a mitochondrial carrier protein during infection. PLoS Pathog. 8, e1002459. 30. Dolezal, P., Likic, V., Tachezy, J. & Lithgow, T. (2006). Evolution of the molecular machines for protein import into mitochondria. Science, 313, 314–318. 31. Herrmann, J. M. (2003). Converting bacteria to organelles: evolution of mitochondrial protein sorting. Trends Microbiol. 11, 74–79. 32. Embley, T. M. & Martin, W. (2006). Eukaryotic evolution, changes and challenges. Nature, 440, 623–630. 33. Hell, K., Herrmann, J., Pratje, E., Neupert, W. & Stuart, R. A. (1997). Oxa1p mediates the export of the N- and C-termini of pCoxII from the mitochondrial matrix to the intermembrane space. FEBS Lett. 418, 367–370. 34. Funes, S., Nargang, F. E., Neupert, W. & Herrmann, J. M. (2004). The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family. Mol. Biol. Cell, 15, 1853–1861. 35. Osman, C., Haag, M., Potting, C., Rodenfels, J., Dip, P. V., Wieland, F. T. et al. (2009). The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria. J. Cell Biol. 184, 583–596. 36. Nebauer, R., Schuiki, I., Kulterer, B., Trajanoski, Z. & Daum, G. (2007). The phosphatidylethanolamine level of yeast mitochondria is affected by the mitochondrial components Oxa1p and Yme1p. FEBS J. 274, 6180–6190. 37. Pfanner, N. & Neupert, W. (1985). Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 4, 2819–2825. 38. Bauer, M., Behrens, M., Esser, K., Michaelis, G. & Pratje, E. (1994). PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast. Mol. Gen. Genet. 245, 272–278. 39. Hell, K., Neupert, W. & Stuart, R. A. (2001). Oxa1p acts as a general membrane insertion machinery for
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proteins encoded by mitochondrial DNA. EMBO J. 20, 1281–1288. Lemaire, C., Guibet-Grandmougin, F., Angles, D., Dujardin, G. & Bonnefoy, N. (2004). A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations. J. Biol. Chem. 279, 47464–47472. Funes, S. & Herrmann, J. M. (2007). Analysis of mitochondrial protein synthesis in yeast. Methods Mol. Biol. 372, 255–263. Nunnari, J., Fox, D. & Walter, P. (1993). A mitochondrial protease with two catalyticsubunits of nonoverlapping specificities. Science, 262, 1997–2004. Käser, M., Kambacheld, M., Kisters-Woike, B. & Langer, T. (2003). Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease. J. Biol. Chem. 278, 46414–46423. Geissler, A., Krimmer, T., Bomer, U., Guiard, B., Rassow, J. & Pfanner, N. (2000). Membrane potentialdriven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsidependence of translocation of the matrix-targeting sequence. Mol. Biol. Cell, 11, 3977–3991. Sikorski, R. S. & Hieter, P. (1989). A system of shuttle vectors and host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122, 19–27. Altmann, K., Dürr, M. & Westermann, B. (2007). Saccharomyces cerevisiae as a model organism to study mitochondrial biology. In Mitochondria Practical Protocols (Leister, D. & Herrmann, J. M., eds), Vol. 372, pp. 81–90. Humana Press, Totowa, NJ. Tjaden, J., Schwoppe, C., Mohlmann, T., Quick, P. W. & Neuhaus, H. E. (1998). Expression of a plastidic ATP/ADP transporter gene in Escherichia coli leads to a functional adenine nucleotide transport system in the bacterial cytoplasmic membrane. J. Biol. Chem. 273, 9630–9636. Mokranjac, D. & Neupert, W. (2007). Protein import into isolated mitochondria. In Mitochondria Practical protocols (Leister, D. & Herrmann, J. M., eds), Vol. 372, pp. 277–286. Humana Press, Totowa, NJ.
http://dx.doi.org/10.1016/j.jmb.2012.07.018 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria Markus Hildenbeutel 1 , Melanie Theis 1 , Melanie Geier 2 , Ilka Haferkamp 2 , H. Ekkehard Neuhaus 2 , Johannes M. Herrmann 1 ⁎ and Martin Ott 3 1
Division of Cell Biology, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany Division of Plant Physiology, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany 3 Research Group Membrane Biogenesis, University of Kaiserslautern, Erwin‐Schrödinger Strasse 13, 67663 Kaiserslautern, Germany 2
Received 4 June 2012; received in revised form 18 July 2012; accepted 20 July 2012 Available online 27 July 2012 Edited by J. Bowie Keywords: carrier proteins; membrane biogenesis; mitochondria; Oxa1 protein import
Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperaturesensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin. © 2012 Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: [email protected]. Present addresses: M. Hildenbeutel, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, SE-106 91 Stockholm, Sweden; M. Ott, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, SE-106 91 Stockholm, Sweden. Abbreviations used: DTT, dithiothreitol; TIM, translocase of the inner membrane; TOM, translocase of the outer membrane.
Introduction Proteins of the inner membrane of mitochondria represent a mosaic of dual genetic origin: The mitochondrial genome encodes a small number of hydrophobic membrane proteins that are integrated into the inner membrane in a co-translational reaction. 1,2 The vast majority of mitochondrial proteins is nuclear encoded and synthesized on cytosolic ribosomes. 3–5
0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.
Oxa1 Facilitates Carrier Biogenesis
Membrane insertion of mitochondrial translation products is facilitated by the inner membrane protein Oxa1. 6–8 Oxa1 is an integral membrane protein with five transmembrane helices and a Cterminal ribosome-binding domain. 1,2 It belongs to a family of proteins with representatives in bacteria (YidC proteins), mitochondria (Oxa1 and Cox18) and plastids (Alb3 and Alb4). Deletion mutants of Oxa1 and YidC show a block in membrane integration of several proteins, suggesting that Oxa1/YidC/Alb3 proteins function as insertases that facilitate membrane integration of transmembrane segments. 6,8–10 In vitro reconstitution experiments of recombinant YidC confirmed its insertase activity. 11,12 In addition to its role as insertase, YidC was proposed to be critical for the folding of some membrane proteins that are initially inserted in an YidC-independent manner. 13,14 A more general role of YidC in the quality control of membrane proteins would be consistent with the severe stress response and upregulation of chaperones and proteases in YidC-depleted cells that were recently observed in proteome-wide analyses. 15–17 However, direct evidence for an insertion-independent folding function of YidC or Oxa1 is lacking since insertion and folding of membrane proteins are difficult to distinguish experimentally. In addition to its role in co-translational protein insertion, Oxa1 was shown to facilitate membrane integration of some nuclear‐encoded proteins that embark on a so-called conservative sorting pathway. 18–20 These substrates are initially imported into the matrix by the TIM23 (TIM, translocase of the inner membrane of mitochondria) complex, the general transporter for matrix proteins. After full or partial translocation, they insert into the inner membrane in an export-like process. All nuclear‐ encoded Oxa1 substrates identified so far (Oxa1, Cox18, Su9/Foc and Mdl1) have homologs in bacteria and presumably evolved from components that were synthesized in the cytosol of the endosymbiotic progenitors of mitochondria. In contrast to conservatively sorted proteins, members of the mitochondrial carrier family use a completely different import pathway that deviates from the import route of matrix proteins after translocation across the outer membrane. They insert from the intermembrane space into the inner membrane in a process relying on a specific inner membrane translocase, the TIM22 complex. 3,21,22 The carrier family contains about 35 members in yeast and more than 50 in humans. 23,24 Carrier proteins are characterized by six transmembrane domains that are arranged in three repetitive modules that tightly interact to form a channel-like structure. 25 The mitochondria-specific lipid cardiolipin plays an important role in the biogenesis, stability and function of carrier proteins. 26–28 Carrier proteins are a signature family of eukaryotic proteins
591 and developed in the cause of the endosymbiont-toorganelle transition of mitochondrial progenitors to connect metabolic and nucleotide networks within the eukaryotic cell. 29–32 Here, we report that Oxa1 plays an unexpected role in the biogenesis of mitochondrial carrier proteins. Upon depletion of Oxa1, carrier proteins are imported into mitochondria with reduced efficiency and accumulate to significantly reduced steady‐state levels. These observations suggest that Oxa1 plays a more general role in inner membrane biogenesis than previously proposed.
Results and Discussion Deletion of Oxa1 leads to reduced levels of the ADP/ATP carrier Oxa1 is known to mediate the insertion of mitochondrially encoded and conservatively sorted inner membrane proteins. 8,18,33,34 Hence, Oxa1 deletion mutants (Δoxa1) show reduced levels of components of respiratory chain complexes (Fig. 1a, Cox2, Cor2, Rip1 and Shy1). Moreover, proteins with conservatively sorted domains rely on Oxa1 and are reduced in the Δoxa1 mitochondria (Fig. 1a, Atm1). 18 In contrast, proteins of the outer membrane, the intermembrane space and the matrix and most proteins of the inner membrane are not affected in Δoxa1 strains (Fig. 1a, Tom70, Mrpl40, Phb2, Yme1, TIM23 and TIM22). Surprisingly, we repeatedly observed significantly reduced levels of ATP/ADP carrier (Fig. 1a, Aac2) in Δoxa1 strains, which was unexpected since carrier proteins reach the inner membrane after insertion from the intermembrane space by the TIM22 translocase. A relevance of Oxa1 for TIM22-mediated protein biogenesis was not reported so far. To further analyze the relevance of Oxa1 for biogenesis of the ATP/ADP carrier, we directly measured the efficiency of ADP transport into wild type and Δoxa1 mitochondria (Fig. 1b). To this end, we incubated de-energized mitochondria from wild type and Δoxa1 cells with 32P-labeled ADP and measured uptake of the radioactive nucleotide. Compared to wild type, ADP uptake was considerably reduced in Δoxa1 mitochondria. Hence, the absence of Oxa1 prevents the generation of normal ATP/ADP carrier levels in mitochondria and, consequently, efficient nucleotide transport. It should be noted that the absence of respiratory chain complexes in Δoxa1 mitochondria leads to a severely reduced membrane potential in this mutant (Fig. 1c). Thus, it remains possible that the pronounced effect of the Oxa1 deletion on carrier accumulation is an indirect effect due to the decreased mitochondrial membrane potential or due to an altered lipid composition in Δoxa1
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(a)
(b)
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Fig. 1. Aac2 levels are reduced in Δoxa1 mitochondria. (a) Mitochondria were isolated from W303 wild type (wt) and an Oxa1 deletion strain (Δoxa1). Protein levels were analyzed by Western blotting using the indicated antibodies. Diminished steady‐state levels of proteins are depicted by arrowheads. IM, inner membrane; OM, outer membrane. (b) Δoxa1 mitochondria show considerably reduced uptake of radioactively labeled [α 32P]ADP. Values for carrierindependent ADP uptake in the presence of atractyloside and bongkrekic acid were subtracted. Shown are mean values of three independent experiments. (c) Δoxa1 mitochondria show a strongly reduced membrane potential. Freshly isolated mitochondria were energized by addition of 2 mM NADH before the membrane potential was analyzed using the dye JC-1. The membrane potential was dissipated again by addition of 25 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Shown are mean values of three measurements. (d and e) The Aac2 levels in Δoxa1 mitochondria are lower than that in mitochondria of a Δcrd1 mutant or of other respiration-deficient strains. Equal amounts of isolated mitochondria of the indicated strains were loaded on SDS gels. For (e), levels of Aac2 were quantified by Western blotting.
mitochondria. 35,36 However, the levels of Aac2 in Δoxa1 mitochondria are lower than those in mitochondria lacking the cardiolipin synthase Crd1 (Fig. 1d) or in mitochondria of other respiration‐ deficient strains (Fig. 1e). Surprisingly, even rho 0 strains in which mitochondrial genomes and, hence, functional enzymes of the respiratory chain are completely absent contain higher levels of Aac2 than Δoxa1 mitochondria.
A temperature-sensitive allele allows the specific depletion of Oxa1 The low membrane potential of Δoxa1 mitochondria makes it difficult to exclude indirect effects on the biogenesis of carrier proteins, which relies on the energization of the inner membrane. 37 In the past, the use of temperature-sensitive Oxa1 variants proved to be a powerful strategy to circumvent this
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problem. 18,38–40 In the oxa1 alleles used so far, incubation at 37 °C inactivated Oxa1 at least partially allowing to analyze its relevance in the context of an otherwise unaltered organelle. Unfortunately, a residual activity of the heat-treated Oxa1 protein cannot be excluded in these strains. To this end, we searched for novel temperature-sensitive oxa1 mu-
tants. We identified an Oxa1 point mutant in which a conserved histidine residue in position 121 was replaced by a leucine residue (Fig. 2a). This mutation provoked a clear-cut temperature-sensitive growth phenotype on non-fermentable carbon sources (Fig. 2b). This respiration deficiency was due to a selective loss of Oxa1 upon incubation at 37 °C,
(a)
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Fig. 2. An H121L mutant of Oxa1 leads to a temperature-sensitive phenotype. (a) A section of an alignment of Oxa1 sequences is presented to show the conservation of the histidine 121 residue. Sequences were used of S. cerevisiae, Kluyveromyces lactis, Schizosaccharomyces pombe, Mus musculus and Homo sapiens. Invariant residues are highlighted. (b) Wild type and Oxa1ts cells were grown to log phase. We dropped 10‐fold serial dilutions on fermentative (glucose) or non-fermentative (glycerol) medium. The plates were incubated at 25 or 37 °C as indicated. (c) Incubation of Oxa1ts cells leads to the depletion of Oxa1. Cells were grown to log phase at 25 °C, washed and further incubated at either 25 or 37 °C for the times indicated. The levels of Oxa1 and Tom70 in whole cell extracts were analyzed by Western blotting. (d) Mitochondria were incubated from wild type or Oxa1ts mutants grown at 25 °C. Following incubation at 25 or 37 °C for the times indicated, we assessed the levels of Oxa1 and Mrp20 by Western blotting. (e) Depletion of Oxa1 prevents maturation of newly synthesized Cox2 precursor (pre-Cox2). Translation products were labeled with [ 35S]methionine in isolated mitochondria 41 for the indicated time periods, separated by SDS-PAGE and detected by autoradiography. Cyt b, cytochrome b.
Oxa1 Facilitates Carrier Biogenesis
594 which was observed when either cells or isolated mitochondria of the oxa1ts strain were incubated at 37 °C (Fig. 2c and d). To analyze the activity of Oxa1 in the Oxa1ts mutant more directly, we grew yeast cells of the wild type or the Oxa1ts mutant at 25 °C and shifted the cultures for 3 h to 25 or 37 °C prior to the preparation of mitochondria. Then, we followed the processing of the mitochondrially encoded protein Cox2 after synthesis in isolated mitochondria (Fig. 2e). Following its insertion, the leader peptide of Cox2 precursor (pre-Cox2) is cleaved by Imp1/Imp2 protease in the intermembrane space. 42 In the absence of Oxa1, membrane insertion of pre-Cox2 is strongly reduced. 8,33 Pre-Cox2 was efficiently matured in Oxa1 and Oxa1ts mitochondria at permissive conditions (Fig. 2e). In contrast, nonprocessed pre-Cox2 accumulated in Oxa1-depleted mitochondria, indicating the absence of Oxa1 activity in these mitochondria. Hence, the Oxa1ts mutant identified in this study allows switching off Oxa1 activity by heat treatment. Depletion of Oxa1 in the Oxa1ts mutant does not influence the membrane potential Next, we directly assessed the protein levels in mitochondria isolated from wild type or Oxa1ts cells after preincubation for 3 h at permissive or restrictive conditions (Fig. 3a). Noteworthy, no Oxa1 could be detected in mitochondria isolated from heatexposed Oxa1ts cells, indicating the efficient deple-
(a)
tion of the Oxa1 mutant protein at 37 °C. Misfolded Oxa1 was shown to be unstable and to be rapidly degraded by mitochondrial proteases. 40,43 Importantly, at these early stages of Oxa1 depletion, respiratory chain components and other mitochondrial proteins were still present at unaltered levels (Fig. 3a). Moreover, this heat treatment did not considerably reduce the membrane potential of these mitochondria, indicating that the inner membrane is still properly energized (Fig. 3b). We next tested the activity of Oxa1-depleted mitochondria in the uptake of 32P-labeled ADP. Similar amounts of ADP were taken up independent of the presence or absence of Oxa1 (Fig. 3c), showing that the chosen regime to deplete Oxa1 does not impair ATP/ADP carrier activity. Taken together, these data show that mitochondria isolated from this Oxa1ts strain at 37 °C specifically lack Oxa1 but contain a functional respiratory chain and an intact inner membrane. Efficient import of carrier proteins depends on the presence of Oxa1 Mitochondria of the heat-treated Oxa1ts mutant allowed us to investigate whether the biogenesis of carrier proteins is affected by the absence of Oxa1. To this end, we followed the import of in vitro synthesized, radioactively labeled precursor proteins into isolated mitochondria (Fig. 4). As expected, matrix proteins such as the ATPase subunit F1β and
(b)
(c)
Fig. 3. Heat exposure of Oxa1ts cells leads to rapid depletion of Oxa1 but does not impair the energization of the inner membrane. (a) Degradation of Oxa1 in the Oxa1ts strain has no effect on the levels of other inner membrane proteins. Yeast cells were grown at 25 °C. Prior to isolation of mitochondria, yeast cells were either kept at 25 °C or shifted to 37 °C for 3 h. Mitochondria were analyzed by Western blotting using the antibodies indicated. (b) The membrane potential in the Oxa1ts mitochondria was analyzed as in Fig. 1c. (c) ADP uptake efficiency was measured as described in Fig. 1b in Oxa1ts mitochondria that were pretreated or not pretreated at 37 °C to induce Oxa1 degradation.
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(a)
(b)
(c)
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Su9 1-69-DHFR were efficiently imported, irrespective of the presence or absence of Oxa1 (Fig. 4a). These results confirmed the intactness of the mitochondrial inner membrane because import of matrix
Fig. 4. Efficient import of members of the mitochondrial carrier family depends on Oxa1. Mitochondria of the indicated strains were incubated for different time periods with different radiolabeled preproteins. Samples were treated with proteinase K and analyzed by autoradiography. The signals were quantified and corrected for the import rates of the matrix protein F1β. Preproteins used were the matrix proteins F1β and Su9 1-69DHFR (a), Aac2 of S. cerevisiae (b), Aac2 of N. crassa (c), the phosphate carrier Pic2 (d) and the dicarboxylate carrier Dic1 of S. cerevisiae (e).
proteins via the TIM23 pathway relies on a membrane potential. Next, we tested whether import of Aac2 into these mitochondria occurs normally. Aac2 was efficiently
Oxa1 Facilitates Carrier Biogenesis
596 imported into mitochondria containing Oxa1 but only with reduced efficiency into mitochondria lacking Oxa1 (Fig. 4b). This dependency on Oxa1 for import was not restricted to yeast Aac2. When Aac2 from the filamentous fungus Neurospora crassa was imported into yeast mitochondria, efficiency of import was decreased as well (Fig. 4c). Moreover, also the import of other members of the mitochondrial carrier family such as the phosphate carrier Pic2 and the dicarboxylate carrier Dic1 was strongly reduced in the absence of Oxa1 (Fig. 4d and e). To exclude indirect effects of slightly reduced levels of the mitochondrial membrane potential, we
performed co-import experiments of a member of the mitochondrial carrier family and F1β into isolated mitochondria in the same reaction. The import of F 1 β requires a robust membrane potential 44 making F1β a very good control for the energy state of the inner membrane. As shown in Fig. 5a and b, the amounts of imported F1β were comparable in the absence or presence of Oxa1, whereas Aac2 from Saccharomyces cerevisiae and N. crassa as well as Pic2 and Dic1 required Oxa1 for efficient import. We therefore concluded that efficient mitochondrial biogenesis of carrier proteins depends on Oxa1. Nevertheless, even in the absence
(a)
(b)
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Fig. 5. Oxa1 depletion specifically impairs the biogenesis of carrier proteins. (a) Mitochondrial carrier proteins and the nuclear‐encoded ATPase subunit F1β were simultaneously incubated with isolated mitochondria for 15 min. Non-imported protein was degraded by proteinase K treatment. Samples were analyzed by autoradiography. (b) The experiment shown in (a) was repeated three times and the signals were quantified. The values indicate the ratios of carrier import relative to the import efficiencies observed for F1β. (c) Aac2 reaches a membrane-embedded location after import into Oxa1ts mitochondria. Aac2 was imported for 30 min into mitochondria of mock- or heat-treated cells. We used a relatively long import time here in order to accumulate significant amounts of Aac2 also in Oxa1-depleted mitochondria. Mitochondria were reisolated and either directly loaded onto the SDS gel (T, total) or separated into a soluble (S) and membrane pellet (P) fraction by carbonate treatment. The samples were analyzed by autoradiography (Aac2) and Western blotting using antibodies against the membrane protein Cox2 and the soluble protein Mprl40. (d) Model for carrier biogenesis. At this stage, it remains unclear whether Oxa1 plays a direct (left) or indirect (right) role during carrier biogenesis.
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of Oxa1, Aac2 finally reached the inner membrane and, after prolonged import periods, could be identified in a carbonate-resistant fraction together with integral membrane proteins such as Cox2 (Fig. 5c).
yeast cultures were either kept at 25 °C or shifted to 37 °C for 3 h in the presence of 20 μM cycloheximide. Mitochondria were isolated as previously described. 46
Conclusion
Radioactively labeled [α 32 P]ADP was synthesized e n z y m a t i c a l l y u s i n g h e xok i n a s e a s d e s c rib ed previously. 47 For ADP uptake measurements, freshly isolated mitochondria were added to 100 μl of transport buffer [20 mM Hepes–KOH (pH 7.0), 15 mM KPi (pH 7.0), 80 mM KCl and 0.6 M sorbitol] containing [α 32P]ADP (Hartmann analytics). Reactions were incubated for 5 s before mitochondria were collected on a filter membrane and extensively washed with ice-cold transport buffer. Radioactivity in each sample was quantified by scintillation counting. To determine background levels, we determined ADP uptake in mitochondria that were pretreated with atractyloside and bongkrekic acid for 5 min to block ATP/ADP carrier activity. Background values were subtracted.
Here, we show that Oxa1 plays a critical role in the biogenesis of mitochondrial carrier proteins. At this stage, it is not clear whether Oxa1 physically interacts with carrier proteins during their import or folding in the inner membrane, and it remains possible that Oxa1 influences the import of carrier proteins only indirectly, for example, by changes in the lipid composition of the mitochondrial membranes (Fig. 5d). However, we regard this as unlikely since the temperature-sensitive mutant that was used in this study, shows wild‐type levels of respiratory chain complexes, of the membrane potential and of the import of matrix proteins even at restrictive conditions. The data shown here indicate that the presence of Oxa1 in the inner membrane is a direct prerequisite for carrier import. Hence, Oxa1 does not only promote the export of proteins from the matrix into the inner membrane but also exhibits a function in the biogenesis of membrane proteins that integrate into the inner membrane via the TIM22 complex. Although we do not know the precise biochemical role of Oxa1 in this process, it appears likely that Oxa1 promotes the folding of newly imported carrier proteins in the inner membrane. A role in the folding of membrane proteins was also proposed for YidC, which was shown to facilitate the biogenesis of proteins that are initially inserted into the bacterial membrane in a YidC-independent process. 13,14,16,17 In summary, we conclude that the substrate spectrum of Oxa1 is presumably much broader than initially expected and not limited to proteins of bacterial origin.
Materials and Methods Yeast strains and growth media All strains used in this study were isogenic to the wild‐ type strain W303. OXA1 was deleted with a HIS3 selection cassette. Oxa1ts strains were derived from a Δoxa1 strain transformed with the plasmid pRS314 45 carrying the endogenous Oxa1 promoter and the temperature-sensitive Oxa1 allele with a H121L mutation. All yeast cultures were grown at 25 °C in lactate medium [0.3% yeast extract, 0.1% KH2PO4, 0.1% NH4Cl, 0.05% CaCl2⋅H2O, 0.05% NaCl, 0.06% MgSO4⋅H2O, 0.0003% FeCl3 and 2% lactate (pH 5.5) (adjust with KOH)] or YP medium (1% yeast extract and 2% peptone) supplemented with either 2% glucose, galactose or glycerol. For the isolation of mitochondria,
Uptake of radioactively labeled ADP into isolated mitochondria
Membrane potential measurements For membrane potential measurements, freshly isolated mitochondria were diluted to an OD595 of 0.25 in a total of 400 μl transport buffer. Mitochondria were energized by addition of 2 mM NADH and incubation of the reaction at 30 °C for 30 s. Upon addition of JC-1 (Calbiochem) to a final concentration of 0.5 μg/ml, the increase in fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 590 nm for 330 s. To dissipate the membrane potential, we added 25 μM carbonyl cyanide m-chlorophenyl hydrazone, and we monitored the decrease in fluorescence for additional 90 s. Import of in vitro synthesized preproteins into isolated mitochondria Proteins were synthesized in reticulocyte lysate as previously described. 48 Per reaction, 40 μg of mitochondria were diluted in 40 μl of import buffer [50 mM Hepes– KOH (pH 7.4), 0.6 M sorbitol, 80 mM KCl, 10 mM Mg acetate, 2 mM KPi and 1 mM MnCl2] in the presence of 2 mM ATP and 2 mM NADH. Import reactions were started by addition of 2 μl of preprotein-containing reticulocyte lysate. Import reactions were stopped by 10‐ fold dilution in ice‐cold SH (sorbitol–Hepes) buffer. Nonimported proteins were removed by addition of proteinase K (100 μg/ml) and incubation for 30 min on ice. Protease treatment was stopped by addition of 1.5 mM PMSF. Mitochondria were isolated by centrifugation and washed three times with SH–KCl buffer [20 mM Hepes– KOH (pH 7.4), 0.6 M sorbitol and 80 mM KCl].
Acknowledgements We thank Jan Riemer for critical reading of the manuscript. We are grateful to Sabine Knaus and
Oxa1 Facilitates Carrier Biogenesis
598 Andrea Trinkaus for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 530 and FOR 967), the Landesstiftung für Innovation Rheinland-Pfalz and the Landesforschungsschwerpunkt Membrantransport.
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