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Report |
Address correspondence to Trevor Lithgow, Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Melbourne 3010, Australia. Tel.: 61-3-8344-4131. Fax: 61-3-9348-2251. email: t.lithgow{at}unimelb.edu.au
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Abstract |
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Key Words: endosymbiont theory; membrane biogenesis; ß-barrel protein; mitochondria; protein import
Abbreviations used in this paper: TOM, translocase in the outer membrane of mitochondria; VDAC, voltage-dependent anion channel.
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Introduction |
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Mitochondrial biogenesis now relies on outer membrane proteins, including subunits of the translocase in the outer membrane of mitochondria (TOM) complex (Neupert, 1997; Voos et al., 1999; Gabriel et al., 2001), components of the mitochondrial fission machinery (Griparic and van der Bliek, 2001; Shaw and Nunnari, 2002), the voltage-dependent anion channel (VDAC) pore that allows ATP/ADP diffusion across the outer membrane (Mannella, 1998; Bay and Court, 2002), and, in metazoans, components from the Bcl-2 family of proteins that regulate the mitochondrial events of programmed cell death (Adams and Cory, 2001; Tsujimoto, 2003). Although it is clear that these nucleus-encoded proteins have helped enslave mitochondria, allowing nuclear genes to control the growth, division, and death of the organelle, a perplexing question has been how these proteins were inserted and assembled in the outer membrane of the endosymbiotic bacteria early in evolution.
Protein insertion into a membrane requires disordering of the lipid bilayer to accommodate a cylinder of polypeptide either in the form of an -helix or a barrel formed of ß-strands. Although little is known about the insertion of proteins into the outer membrane of bacteria (Kleinschmidt, 2003), recent work suggests that the protein Omp85 is an essential component for outer membrane biogenesis in the gram-negative bacterium Neisseria meningitidis. The gene is positioned in the lipopolysaccharide biosynthetic operon in gram-negative bacteria (Genevrois et al., 2003; Voulhoux et al., 2003), and Omp85 is required for effective insertion of lipids (Genevrois et al., 2003) and integral proteins into the outer membrane of N. meningitidis (Voulhoux et al., 2003). The insertion of bacterial outer membrane proteins is also assisted by soluble chaperones that can dock transiently with the membrane surface (Tamm et al., 2001; Kleinschmidt, 2003).
Comparative sequence analysis suggests that Omp85 homologues are found in all bacteria that possess an outer membrane and that the Omp85 family is represented in the outer membrane of mitochondria in eukaryotes from plants to humans. In the yeast Saccharomyces cerevisiae, Omp85 is essential for cell viability. Conditional omp85 yeast mutants are compromised in the insertion and assembly of integral outer membrane proteins, including the VDAC pore and components of the TOM complex. We propose the Omp85 family of proteins as the core component for biogenesis of outer membranes of bacteria and mitochondria, and that Omp85 was required in the earliest stage of converting endosymbiotic bacteria to organelles.
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Results and discussion |
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Homologues of Omp85 occur in fungi, plants, and animals (including humans). These eukaryotic Omp85 family members are truncated, with a short NH2-terminal domain, but the conserved COOH-terminal domain of the protein also predicts strongly to have 12 membrane-embedded ß-strands. Across the prokaryotic and eukaryotic members of the Omp85 family, there is strong sequence conservation in the regions predicting as ß-strands, with variable intervening sequences that might correspond to interstrand loops (Fig. 1 B). Phylogenetic analyses of Omp85 show that these eukaryotic homologues strongly grouped together and cluster within the proteobacteria, grouping most strongly with the -proteobacteria, the progenitor lineage for mitochondria (Fig. 1 A). This is consistent with this molecule deriving from the original mitochondrial endosymbiont (Emelyanov, 2003). A second class of homologues in eukaryotes, the plastid protein translocator Toc75, does not group within the proteobacteria but rather clusters with the cyanobacteria. Cyanobacteria gave rise to plastids in plants and algae, and, therefore, Toc75 most likely had an independent origin in eukaryotes.
Yeast Omp85 is an integral protein in the mitochondrial outer membrane
Antibodies were raised to the yeast Omp85, affinity purified on the recombinant protein, and used to decorate thin sections of cryo-preserved yeast cells. Gold particles denote the presence of Omp85 on the surface of mitochondria (Fig. 2 A). Quantitation of gold labeling in 50 randomly selected cell sections showed 99 particles on or within 50 nm of the mitochondrial outer membrane, with a signal/noise ratio (West et al., 1998) of >66:1. Subcellular fractionation confirms that Omp85 is a mitochondrial protein; immunoblots decorated with specific antibodies show enrichment of both Omp85 and the outer membrane pore VDAC in purified mitochondria (Fig. 2 B).
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Omp85 performs an essential function in mitochondria
The OMP85 gene is essential for viability (unpublished data). A plasmid carrying the open reading frame for Omp85 could rescue the omp85 cells, allowing wild-type growth on fermentable (glucose) and nonfermentable (lactate) carbon sources (unpublished data). This strain, IGY001, was used as a basis to select for conditional omp85 mutants.
Low fidelity PCR was undertaken and several hundred mutants were assayed for conditional growth defects. 20 were selected for further study (Table I). Mutants displaying a range of phenotypes were isolated, suggesting that pleiotropic defects can arise from debilitated function of Omp85. Many of the alleles display growth defects on the nonfermentable carbon source lactate, where growth depends on robust mitochondrial function.
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Omp85 mediates protein insertion and assembly in the mitochondrial outer membrane
To assay the kinetics of mitochondrial protein insertion and assembly, mitochondria were isolated from the omp85-94 mutant and incubated with radiolabeled Tom40 or VDAC (Fig. 3). Tom40 is assembled into the hetero-oligomeric TOM complex through a series of transient assembly intermediates, visualized in time course experiments analyzed by BN-PAGE (Model et al., 2001; Wiedemann et al., 2003). The omp85-94 mutant shows a twofold defect in the kinetics of Tom40 assembly into the TOM complex (Fig. 3 A). Similarly, assembly of VDAC into the trimeric native state can be analyzed by BN-PAGE (Krimmer et al., 2001) and is achieved at 50% the wild-type rate in the omp85-94 mutant (Fig. 3 B). Insertion of VDAC into the outer membrane, as judged by accumulation of a protease-resistant form, is only marginally compromised in the mutants (Fig. 3 B, SDS-PAGE). The protein translocation defect in the omp85-94 mutants is selective for outer membrane proteins, with import of the matrix-located protein Su9-DHFR proceeding at wild-type rates in the omp85-94 mitochondria (Fig. 3 C).
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The assembly defects seen in omp85 mutants are reminiscent of the phenotypes observed in mitochondria lacking Mas37 (Wiedemann et al., 2003). Mas37 is a peripheral component of an integral membrane complex operationally defined as the SAM complex (for sorting and assembly machinery). Using affinity-tagged Mas37, the SAM complex can be purified and the integral component shown to be Omp85 (Kozjak et al., 2003). In bacteria, the soluble factors Skp and SurA dock peripherally to the membrane to assist protein insertion (Tamm et al., 2001; Kleinschmidt, 2003). Comparative sequence analysis has not revealed to us any yeast homologues to these bacterial chaperones, and further work will be required to determine whether Mas37 is the functional equivalent of these chaperones in mitochondria.
We propose that Omp85 acts as an intramembrane molecular chaperone, transiently interacting with newly arrived substrate membrane proteins, assisting them to locally disorder a lipid bilayer and insert amid the lipids. Two distinct architectures are possible for polypeptides to span a lipid bilayer: -helices, a small diameter cylinder of polypeptide, and ß-barrels, where the amide hydrogen bond donors and acceptors complement each other in a ß-sheet wrapped so the edges of the sheet associate to produce a larger diameter cylinder of polypeptide (Schulz, 2002). Inserting either
-helices or ß-barrels of polypeptide into a bilayer is an unfavorable reaction, exacerbated where multiple transmembrane segments have to be assembled and where hydrophilic domains and interstrand loops have to be translocated across the bilayer.
While a detailed structural analysis of the Omp85 family members awaits, we suggest that dynamic interactions with select surface patches of Omp85 might assist the insertion and final folding of other barrels like Tom40 and VDAC, and probably also substrate proteins with -helical transmembrane domains. Crystal structures available for several different ß-barrel proteins show that distinct surface features can be present on the barrels, including an "aromatic girdle" of residues (especially phenylalanines) at the lipid interface and patches of large, flexible side chains interspersed with cavities formed with the placement of small residues like glycine and alanine (Buchanan, 1999; Tamm et al., 2001; Schulz, 2002). Surface features of Omp85 might also assist the insertion of new lipid monomers in line with the roles proposed in bacterial membrane biogenesis.
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Materials and methods |
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Antibody production and immunoelectron microscopy
The open reading frame encoding Omp85 (YNL026w) was cloned into the bacterial expression vector pQE10 and the protein was overproduced in inclusion bodies. Antiserum was prepared from rabbits injected with recombinant Omp85. Wild-type yeast from liquid culture were transferred to 200-nm deep, brass planchettes for immediate high-pressure freezing (Leica). Freeze substitution (Leica) was in anhydrous acetone containing 0.1% uranyl acetate (wt/vol) at -90°C for 76 h. Samples were warmed to -45°C, washed in anhydrous acetone, infiltrated with Lowicryl HM20 resin over 3 d, and polymerized under UV light at -45°C. Thin sections were labeled with affinity-purified anti-Omp85 and stained with aqueous uranyl acetate and lead citrate before viewing on a Philips CM12 transmission electron microscope. Control experiments, omitting the primary antibody, did not specifically label any cellular structures (not depicted).
Yeast growth and mitochondrial isolation
Using PCR-mediated gene disruption (Longtine et al., 1998), a single copy of the YNL026w gene was deleted in diploid yeast to generate the heterozygous strain IGY002. YNL026w is essential for viability in haploid progeny derived from IGY002. IGY002 was transformed with plasmid pYX213 carrying the OMP85 open reading frame under the control of the GAL1 promoter, sporulated, and dissected to generate the omp85Ø strain. IGY002 was alternatively transformed with a plasmid copy of YNL026w under the control of the MET25 promoter, sporulated, and dissected to generate the yeast strain IGY001 (omp85::HIS3, his3, leu2-3,112,
ura3, ade2, p416MET25OMP85::URA3). Conditional omp85 mutants were constructed using low-fidelity PCR to mutate a fragment of DNA corresponding to the YNL026w gene followed by recombination of the mutant allele onto plasmid pRS314 in vivo (Gabriel et al., 2003) after transformation into IGY001. Trp+ transformants were selected at 25°C and screened for growth at 15°C, 25°C, and 37°C on minimal glucose media containing 5-fluoroorotic acid and appropriate growth supplements. Approximately 300 mutants were collected and screened for growth defects.
Mitochondria were isolated from wild-type or mutant yeast cells and characterized as previously described (Gabriel et al., 2003). Mitochondrial proteins were separated by SDS-PAGE or BN-PAGE and analyzed by immunoblotting according to previous methods (Krimmer et al., 2001; Model et al., 2001). Omp85 insertion into mitochondria was assessed according to methods for import of F1ß (the ß-subunit of the F1F0-ATP synthetase) and cytochrome b2 (Cyb2) into isolated mitochondria (Gabriel et al., 2003) and measurements of alkali-resistant insertion of VDAC (Krimmer et al., 2001).
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Acknowledgments |
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This work was supported by a grant from the Australian Research Council (to T. Lithgow) and Australian Postgraduate Research Awards (to I. Gentle and K. Gabriel).
Submitted: 20 October 2003
Accepted: 18 November 2003
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