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Vol. 19, Issue 1, 126-136, January 2008
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Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Submitted August 20, 2007;
Revised October 16, 2007;
Accepted October 19, 2007
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gabriel et al., 2001; Schleiff and Soll, 2005).
The assembly of β-barrel proteins into the mitochondrial outer membrane requires the sorting and assembly machinery (SAM) complex (Pfanner et al., 2004; Paschen et al., 2005). Like other mitochondrial-targeted proteins, β-barrel proteins are first translocated across the outer membrane via the translocase of the outer membrane (TOM) complex, and, in a manner dependent on chaperones in the intermembrane space, they are then passed on to the SAM complex for their final assembly into the outer membrane (Pfanner et al., 2004; Paschen et al., 2005). Studies in yeast have identified key components of the SAM complex: Sam50 (also called Tob55; Kozjak et al., 2003; Paschen et al., 2003; Gentle et al., 2004), Sam35 (also called Tom38 and Tob38; Ishikawa et al., 2004; Milenkovic et al., 2004; Waizenegger et al., 2004), and Sam37 (also called Mas37; Gratzer et al., 1995; Wiedemann et al., 2003). Sam50, the membrane-embedded subunit of the SAM complex, is an essential protein predicted to have a β-barrel topology, and it is related to the Omp85 family of proteins that mediate protein assembly into bacterial outer membranes (Dolezal et al., 2006; Gentle et al., 2004, 2005). The other two subunits, Sam35 and Sam37, are peripheral membrane proteins that are assumed to associate with the outer membrane via direct contact with Sam50 (Kozjak et al., 2003; Milenkovic et al., 2004). The complex formed between Sam50, Sam35, and Sam37 is responsible for the assembly of all β-barrel proteins in yeast, and it is referred to as the SAMcore complex. Mdm10, another β-barrel protein involved in maintaining mitochondrial morphology and distribution (Sogo and Yaffe, 1994; Meisinger et al., 2004), has been shown to interact with the SAMcore complex and to have a specific role in assembling Tom40 into the TOM complex (Meisinger et al., 2004). Mdm10, and perhaps other proteins, form modules that give rise to a SAM supercomplex.
Recent studies show that although the SAMcore complex assists assembly of all β-barrel proteins, additional factors are required to mediate Tom40 assembly into a TOM complex (Ishikawa et al., 2004; Waizenegger et al., 2005). The identification of the SAM complex together with these new components mediating more select aspects of membrane protein assembly, has set the basic framework for a detailed characterization of the mechanisms driving the pathway of β-barrel protein assembly. Recently, it has been shown that the N-terminal domain of Sam50 exposed to the intermembrane space has receptor-like function for β-barrel proteins and may assist translocation of substrates from the trans side of the TOM complex to the SAM complex (Habib et al., 2007). Tom7, a conserved subunit of the TOM complex, mediates the segregation of Mdm10 from its interaction with the SAMcore complex (Meisinger et al., 2006). Mdm10 was very recently shown to associate with the Mdm12/Mmm1 complex, and both Mdm12 and Mmm1 are important for β-barrel protein assembly (Meisinger et al., 2007).
Despite the recent advances, little yet is known about how the two peripheral components of the SAMcore complex, Sam35 and Sam37, function. Their general involvement in constituting the SAMcore complex makes them important in β-barrel protein assembly, but their individual contributions to the function of the SAMcore complex remains unclear. We find a codependent relationship between Sam37 and Sam35. Mutations in Sam35 leads to decreased levels of Sam37, and deletion of Sam37 causes decrease in Sam35 levels. By maintaining the levels of Sam35 in 
sam37 mitochondria, we show that the Sam35-Sam50 is fully capable of assembling β-barrel precursors into their functional complexes. Two yeast mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required in order for the Sam50 subunit to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by the SAM complex. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sam35::His3-MX6) was generated by direct gene replacement with one copy of SAM35 open reading frame (ORF) by using methods described previously (Longtine et al., 1998). NCY0603 (MAT
his3-11 leu2-3 ura3-1 ade2-1 trp1-1 can1-100 sam35::His3-MX6 URA3::YEpSam35) was generated by sporulation and dissection of NCY0601 transformed with YEpSam35. sam37 strain (MAT his3-11 leu2-3 ura3-1 ade2-1 trp1-1 can1-100 sam37::His3-MX6) was from Ian Gentle (Department of Biochemistry and Molecular Biology, University of Melbourne) and was generated as described previously (Longtine et al., 1998). All yeast strains used in this study were derived from W303 background.
Generation of sam35 Random Mutant Library
Conditional alleles of sam35 were generated by low-fidelity polymerase chain reaction (PCR) to mutate a fragment of DNA containing SAM35, followed by recombination of mutant alleles with linearized pRS314 (CEN6, TRP1; Sikorski and Hieter, 1989) in vivo upon transformation into NCY0603 by using similar procedures as described previously (Sikorski and Hieter, 1989; Gabriel et al., 2003). Ura+ Trp+ transformants were selected at 25°C. Plasmids encoding the wild-type copy of SAM35 (YEpSam35) was ejected by selection of transformants on minimal glucose media containing 5-fluroorotic acid and appropriate supplements at 25°C. Approximately 600 transformants were collected and screened for growth defects at 25, 30, and 37°C on rich media containing glucose (YPAD) or glycerol (YPG). Plasmids were isolated from the yeast mutants that showed conditional phenotypes and mutations in the SAM35 ORF confirmed by DNA sequencing. Specificity of the mutant sam35 induced phenotypes were confirmed by isolating plasmids from the conditional mutants, followed by retransforming into NCY0603, plasmid shuffling as described above to eject pRS-SAM35, and growth phenotype was tested at 25, 30, and 37°C on rich media containing glucose (YPAD) or glycerol (YPG).
Plasmid Construction
All plasmid manipulations were carried out using protocols described previously (Sambrook and Russell, 2001). Details of each construct and sequences of all oligonucleotides used are available upon request. In brief, constructs used in the multicopy suppression experiments were generated by PCR amplification of genomic fragments encompassing the gene of interest from yeast genomic DNA and cloned into YEplac181 (LEU2, 2µ). To construct YEpSam35, the genomic fragment containing the SAM35 ORF plus 530-bp upstream and 466-bp downstream sequences was amplified from yeast genomic DNA and cloned into the BamHI, HindIII sites of the multicopy yeast expression vector YEplac195 (URA3, 2 µ). pRS-SAM35 (wild-type control for sam35 mutant alleles) was generated by subcloning the BamHI, HindIII fragment from YEpSam35 into pRS314 (Sikorski and Hieter, 1989).
Growth Assays
Cells were grown in rich media (YPD) or synthetic complete media (with glucose) lacking appropriate amino acids for plasmid selection to mid-logarithmic phase. Cells were diluted to OD600 of 0.04 followed by a series of fivefold dilutions and spotted onto the indicated plates and incubated at the indicated temperatures for 2–5 d.
Isolation of Mitochondria and In Vitro Protein Import
Mutant yeast strains and their corresponding wild-type control strains were grown in parallel in lactate medium at 25°C. Mitochondria were isolated by differential centrifugation as described previously (Daum et al., 1982). For in vitro transcription, pSP65 or pGEM vectors carrying ORFs of mitochondrial precursor proteins were linearized by restriction digest at a unique site downstream of the ORFs and used for in vitro transcription by using SP6 polymerase (Promega, Madison, WI) according to the manufacturer's instructions. Radiolabeled precursor proteins were in vitro translated in rabbit reticulocyte lysates (Promega) in the presence of [35S]methionine/cysteine (MP Biomedicals, Irvine, CA) at 30°C for 30 min before import experiment. For in vitro import, isolated mitochondria (25 µg/time point for SDS-polyacrylamide gel electrophoresis [PAGE]; 50 µg/time point for blue native [BN]-PAGE) were incubated in import buffer (0.6 M sorbitol, 50 mM HEPES, pH 7.4, 2 mM KPi, pH 7.4, 25 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, and 1 mM dithiothreitol) supplemented with 5 mM NADH and 1–4 mM ATP. We added 5% (vol/vol) 35S-labeled precursors to the mitochondria and incubated them at 25°C for the indicated time. Import reactions were stopped by dilution of import reaction in ice-cold import buffer with 100 µM carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) and incubation on ice for matrix and inner membrane-targeted precursors. Proteins not imported into mitochondria were removed by treatment with 50 µg/ml proteinase K for 10 min. Proteinase K digestion was terminated by addition of 1 mM phenylmethylsulfonyl fluoride (PMSF). Mitochondria are isolated, boiled in sample loading buffer, and analyzed by SDS-PAGE. Phosphorimage analysis was carried out using a Typhoon TRIO variable mode imager (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), and quantification of radioactive signal was accomplished using ImageQuant software (GE Healthcare). For assembly assays analyzed by BN-PAGE, import reactions are terminated by incubation on ice without addition of CCCP or proteinase K. Mitochondria are isolated by centrifugation and solubilized as described below.
Blue Native Polyacrylamide Gel Electrophoresis
Mitochondria (50–100 µg of proteins) were solubilized by resuspension in 50 µl of ice-cold digitonin-containing buffer (0.8–1% digitonin, 20 mM Tris-Cl, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, and 1 mM PMSF, pH 7.4) and incubated on ice for 15 min with intermittent vortexing. Insoluble materials were pelleted by centrifugation at 10,000 rpm for 10 min, and the supernatant containing the protein complexes was transferred to a new tube. Loading buffer (13 µl of 5% Coomassie Blue G and 500 mM amino caproic acid in 100 mM bis-Tris, pH 7.0) was added to the supernatant, and the protein complexes were separated by BN-PAGE on a 6–16.5% polyacrylamide gel (Nijtmans et al., 2002; Wittig et al., 2006). For immunoblotting, the protein complexes were transferred onto polyvinylidene difluoride (PVDF) membrane and detected by antibodies using enhanced chemiluminescence methods. For import experiments, the BN gel was dried, and radiosignals were detected by phosphorimage analysis (GE Healthcare).
Confocal Microscopy
Wild-type and mutant strains were grown in synthetic complete media at 30°C with lactate as a carbon source to OD600 
0.8. Cells were stained with MitoTracker Red (Invitrogen, Carlsbad, CA), and mitochondria were visualized using a TCS SP2 imaging system (Leica, Wetzlar, Germany). In each experiment, 100 cells were randomly selected, and their mitochondrial morphology was assessed individually.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sam35/SAM35) diploid yeast lacking one copy of the SAM35 gene were transformed with a control (multicopy) plasmid YEplac195, or the same plasmid containing the SAM35 gene (YEpSam35), or the same plasmid but containing instead the SAM37 gene (YEpSam37). After sporulation, the progeny of meiosis were dissected onto rich medium containing glucose as a carbon source: overexpression of the SAM37 gene cannot compensate for the absence of SAM35 (Figure 1C).
Characterization of sam35-424
To investigate the defects in sam35-424, mitochondria were isolated from sam35 cells transformed with either a plasmid carrying the wild-type SAM35 gene ("SAM35"), or the mutant sam35-424 gene, after growth of the transformed cells at 25°C in lactate medium. Steady-state levels of various mitochondrial proteins as analyzed by immunoblotting showed no difference in any other mitochondrial proteins, including the level of the mutant sam35 protein observed in the sam35-424 strain (Figure 2A). The steady-state level of assembled TOM complex in sam35-424 is similar to wild type (Figure 2B), but immunoblotting from the same BN-PAGE gels with anti-Sam50 antibodies revealed defects in the assembly state of the SAM complex (Figure 2C). An additional complex of 80 kDa, containing Sam50, is absent in the wild-type control, but it is observed in sam35-424, suggesting that the SAM complex in sam35-424 is less stable and more sensitive to detergent treatment. This 80-kDa complex does not contain Sam35, as judged from immunoblotting by using anti-Sam35 antibodies (Figure 2C).
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To assess the assembly kinetics of Tom40 into the TOM complex, 35S-labeled Tom40 was imported and analyzed by BN-PAGE (Figure 2E). A major defect is seen in the total amount of Tom40 taken up by mutant mitochondria, with some accumulation of Tom40 at the 100-kDa assembly intermediate II, where Tom40 is in contact with Tom5 and/or Tom6 (Model et al., 2001; Wiedemann et al., 2003; Figure 2E). Whether the 100-kDa intermediate is found free in the outer membrane or represents a species that is more readily solubilized with digitonin during the assembly of Tom40 into the TOM complex by the SAM complex remains to be determined (see Discussion).
Multicopy Suppression of sam35-424 Cells Suggests Sam37 Acts Downstream of Sam35
Mitochondria were isolated from sam35 cells transformed with plasmids either carrying the wild-type SAM35, or sam35-424, or sam35-424 and overexpressing SAM37, and immunoblots confirmed overexpression of Sam37 (Figure 3A). Because the SAM complex of sam35-424 is destabilized (Figure 2C), we investigated whether overexpression of Sam37 in sam35-424 resulted in stabilization of the SAM complex, which could in turn enable more efficient assembly of Tom40 into the TOM complex and the assembly of other β-barrel proteins, explaining the suppressed growth phenotype of sam35-424. To test this, mitochondria from sam35-424 and sam35-424 overexpressing Sam37 were solubilized in digitonin-containing buffer, and protein complexes were analyzed by BN-PAGE. Anti-Sam50 was used to immunoblot for the SAM complexes (Figure 3B). Overexpression of Sam37 does not stabilize the SAM complex in sam35-424, because the SAM complex in sam35-424 overexpressing Sam37 is similar to sam35-424 mitochondria alone.
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sam35 cells transformed with plasmids either carrying the wild-type SAM35, or sam35-424, or sam35-424 and overexpressing SAM37, but no improvement of the amount of porin or Tom40 imported into sam35-424 when Sam37 was overexpressed (Figure 3C). Because the imported Tom40 shown in Figure 3C represents Tom40 molecules that are in a protease inaccessible and membrane-protected environment, it does not discriminate between forms of Tom40 in assembly intermediates from those that are properly assembled into the TOM complex, as both are protease insensitive (Model et al., 2001). To address this, we analyzed the import of Tom40 by BN-PAGE (Figure 3D). Consistent with the results in Figure 2E, where the overall signal from the radiolabeled Tom40 is highly reduced in both sam35-424 and when it is overexpressing Sam37, a clear difference can be seen in the distribution of Tom40 on BN-PAGE. Overexpression of Sam37 promotes the assembly of Tom40 molecules into the TOM complex, without improving the capacity of sam35-424 mitochondria to import more Tom40.
Overexpression of Sam37 Stabilizes the SAM Complex in sam35-409, and Steady-State Levels of Sam35 and Sam37 Are Codependent
The sam35-409 mutant strain is also temperature sensitive, and, like sam35-424, its phenotype is suppressed by overexpression of SAM37. However, the mechanism of suppression of the sam35-409 phenotype by Sam37 is distinct from that seen in sam35-424. Mitochondria were isolated from sam35 cells transformed with plasmids either carrying the wild-type SAM35, or sam35-409, or sam35-409 and overexpressing SAM37. Immunoblotting for Sam35 by using two independent polyclonal sera raised against Sam35 shows that the steady-state level of the mutant protein in sam35-409 is significantly reduced, leading to a moderately reduced level of Sam50 and a highly reduced level of Sam37 (Figure 4A). The levels of outer membrane protein Tom70 and Tom20, inner membrane protein Tim23, and matrix protein mtHsp70 remain similar to wild type. By overexpressing Sam37 in sam35-409, the levels of the mutant sam35 protein and Sam50 are largely restored (Figure 4A). The steady-state level of the SAM complex, too, is restored as judged by immunoblots from BN-PAGE (Figure 4B). Concomitantly, the steady-state level of assembled TOM complex is like wild type in mitochondria from sam35-409 cells, provided SAM37 is overexpressed (Figure 4C).
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Decreased Levels of Sam35 Contribute to Phenotypes of sam37 Cells
Given the apparent codependence on Sam37 for the function of Sam35, we asked whether overexpression of Sam35 in sam37 cells suppresses any aspects of the phenotype of sam37 cells. The growth of wild-type (W303), sam37, and sam37 cells overexpressing Sam35 was compared. The sam37 mutants are temperature sensitive (Figure 6A; Gratzer et al., 1995; Meisinger et al., 2007). Overexpression of Sam35 in sam37 cells can suppress the growth defects at 25 and 30°C, but not the lethality at 37°C (Figure 6A). Because sam37 cells also display a mitochondrial morphology defect (Meisinger et al., 2004), we asked whether overexpression of Sam35 can suppress this defect as well. Confocal microscopy was used to compare mitochondrial morphology from 100 cells randomly selected from each of wild-type and mutant strains grown in lactate medium at 30°C. In wild-type cells, almost all cells contain the normal reticulated network of mitochondria (Figure 6B), whereas almost all sam37 cells contained mitochondria with aberrant, aggregated mitochondria. Overexpression of Sam35 in sam37 cells restored 90% of the cells to wild-type mitochondrial morphology (Figure 6B).
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sam37 cells, mitochondria were isolated from wild-type, sam37, and sam37 cells overexpressing Sam35 from lactate medium at 25°C. Mitochondria were analyzed by SDS-PAGE and immunoblotted for SAM complex components and various mitochondrial proteins (Figure 6C). The levels of the TOM complex receptors (Tom70 and Tom20), the core subunit of the inner membrane TIM23 translocase (Tim23), and matrix proteins (mtHsp70, F1β, and Mdj1) are very similar between wild-type, sam37, and sam37 cells overexpressing Sam35. The level of Sam35, however, is dramatically reduced in sam37 mitochondria, and the level of Sam50 is moderately reduced. The level of Tom40 is also reduced in sam37 mitochondria, but we did not detect a reduction in the levels of porin. By overexpressing Sam35 in sam37 cells, the level of Sam35 restored, and it also largely restored the levels of Sam50 and Tom40 (Figure 6C).
Consistent with the decreased Tom40 levels in sam37 mitochondria, the TOM complex is also diminished (Figure 6D). By restoring the levels of Sam35 in sam37 mitochondria via overexpression of Sam35, the steady-state levels of the TOM complex is restored (Figure 6D). An improved import of Su9-DHFR and porin across the outer membrane when Sam35 is overexpressed in sam37 mitochondria (Figure 6E) is consistent with the observed increase in the steady-state level of TOM complex.
Sam35–Sam50 Complex Is the Functional Core Module of the SAM Complex
Because loss of Sam37 leads to reduced levels of Sam35, we used BN-PAGE to analyze this effect at the level of the SAM complex (Figure 7A). Immunoblotting for Sam50 and Sam35 shows that sam37 mitochondria cannot form the 230-kDa SAM complex but instead forms an 130-kDa complex consisting of Sam50 and Sam35 (SAM'; Figure 7A), as reported previously (Wiedemann et al., 2003; Waizenegger et al., 2004), but even this 130-kDa complex is present at reduced levels in sam37 mitochondria. Overexpression of Sam35 in sam37 mitochondria significantly stabilized the 130-kDa Sam35–Sam50 complex (Figure 7A).
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sam37 mitochondria where very little 35S-labeled Tom40 precursor is bound, stabilization of the Sam35–Sam50 complex (via overexpression of Sam35) is sufficient to restore high levels of Tom40 precursors bound even in the absence of Sam37. Together, our data suggest that the Sam35–Sam50 complex is sufficient to assemble β-barrel proteins into the outer membrane. The assembly defect in sam37 mitochondria is partly due to the decreased levels of Sam35, and the function of Sam37 is to facilitate release of substrate β-barrels from the SAMcore complex. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In sam35-409 cells the steady-state levels of the mutant Sam35 protein is significantly decreased, leading to a strong decrease in the levels of the SAMcore complex as detected by BN-PAGE. Overexpression of SAM37 in the sam35-409 mutants suppressed the mutant phenotype by maintaining the levels of the mutant sam35-409 subunit in the SAMcore complex. Likewise, sam37 cells can be cured of protein import defects by overexpression of SAM35, which restores a stable SAM' complex (albeit without Sam37). The SAM' complex is fully competent for binding Tom40 substrate. Together, the data presented here suggest an important function of Sam37 is to maintain the stability of Sam35; but an additional, specific function is to affect the release of folded substrate proteins from the SAM complex.
The phenotypes of the sam35 alleles and the mechanism and consequences for Sam37 multicopy suppression of the phenotypes of these alleles can be explained by two potential, related activities for Sam35. In the first, Sam35 is a receptor for Tom40 and other β-barrel proteins, being the first point of contact for substrates entering the SAM complex. However, because substrates enter the SAM complex from the intermembrane space (Model et al., 2001), and Sam35 is exposed on the mitochondrial surface (Ishikawa et al., 2004; Milenkovic et al., 2004; Waizenegger et al., 2004), any "receptor" domain of Sam35 would need to sit exposed to the intermembrane space. Mutations like that in the sam35-424 cells that decrease receptor activity, or a decrease in the level of Sam35 as seen in sam37 cells, would thereby inhibit import of Tom40.
Our data also suggest Sam35 directly assists Sam50 to form the 250-kDa assembly intermediate of Tom40 (or an equivalent substrate:SAM complex for other β-barrel substrates). If the precise function of Sam35 in this process is to assist binding of β-barrel substrates to Sam50, it would explain the subtle effects seen in sam35-424 cells overexpressing Sam37: mitochondria from these cells remain incompetent at binding high levels of Tom40 substrate because of the mutation in Sam35, but they exhibit an increased clearance of the bound Tom40 out of Sam50, provided enough Sam37 is present. We would argue that the sam35-424 mutant sits at a functional tipping point and that Sam37 can influence the structural stability of the mutant Sam35 protein to enhance the release of β-barrel substrates without a gross improvement of the binding capacity seen in the SAM complex.
Does this codependency hold for the metaxins, the putative human counterparts of Sam35 and Sam37? The SAM complex found in human cells is around 300 kDa (Humphries et al., 2005; Kozjak-Pavlovic et al., 2007), and it does not contain metaxins stably bound to it (Kozjak-Pavlovic et al., 2007). Although a proportion of Metaxin 1 and Metaxin 2 are found on the mitochondrial surface, the proteins have also been observed free in the cytosol (Armstrong et al., 1997). Metaxin 1 and Metaxin 2, which share sequence similarity to Sam37 and Sam35, respectively, are found together in a much larger complex of 600 kDa (Kozjak-Pavlovic et al., 2007). Consistent with the codependence of Sam35 and Sam37, RNAi knockdown of the expression levels of Metaxin 2 causes a concomitant decrease in Metaxin 1 levels. Mitochondria from these "metaxin-depleted" cells are defective in assembly of β-barrel proteins into their outer membrane (Kozjak-Pavlovic et al., 2007).
Has Assembly Intermediate II Already Left the SAM Complex?
After import, unfolded Tom40 precursors rapidly bind the SAMcore complex to form an assembly intermediate I that migrates on BN-PAGE at 250 kDa and contains Sam37, Sam35, and Sam50 as well as Tom40 substrate (Model et al., 2001; Paschen et al., 2003; Wiedemann et al., 2003; Ishikawa et al., 2004; Milenkovic et al., 2004; Waizenegger et al., 2004). Under the same solubilization conditions, Tom40 substrates can be seen to move subsequently into a form that contains Tom5 and perhaps other small Tom proteins (Model et al., 2001; Wiedemann et al., 2003), with this intermediate running at 100 kDa on BN-PAGE (Model et al., 2001). No SAM complex subunits comigrate with this solubilized complex. One possibility is that this assembly intermediate has been released from the SAM complex to exist independently in the outer membrane and that other Tom subunits (such as Tom7, Tom22, and Tom20) will be subsequently added, unassisted, to the assembly intermediate to eventually form a mature TOM complex.
However, an alternative possibility is that the 100-kDa assembly intermediate II is still bound to the SAM complex in outer membranes but that the nascent substrate complex is solubilized out of the SAM complex by the lysis conditions used for BN-PAGE. This distinction matters. It offers an explanation to our observation that Sam37 can function as an assembly factor for Tom40 downstream of the 250-kDa intermediate I, by facilitating the progression of Tom40 from the 100-kDa intermediate II to the mature TOM complex. It also helps rationalize observations made of Mdm10: that it mediates the late stage of TOM complex assembly (from 100-kDa intermediate II to mature TOM complex) and that it associates with the SAM complex. The functional interplay between Tom7 and Mdm10 identified recently by Meisinger et al. (2006) is also consistent with this alternative interpretation.
Why Do Mitochondria Need Sam35 and Sam37, When Bacteria Do Not?
The mitochondrial SAM complex is derived from the bacterial Omp85 complex, but, to date, no detailed structural analysis of a mitochondrial β-barrel protein has been completed. Might differences in the mitochondrial β-barrel substrate proteins dictate a need for Sam35 and Sam37? One clear difference exists in the nature of the external environment of the mitochondrial and bacterial outer membranes. Bacterial outer membranes are built on an asymmetric bilayer, with phospholipids confined to the inner leaflet and glycolipids in the outer leaflet (Kamio and Nikaido, 1976). Bacterial β-barrel proteins have elongated interstrand loops that would likely sit within the glycolipid environment: mitochondrial β-barrels have interstrand loops that would be exposed to the cytosol and might be more difficult to fold, require protection from cytoplasmic proteases during the folding process, or both. Both the transient interaction of metaxins in human cells and the constant, sheltering presence of Sam35 and Sam37 in yeast would afford a protective environment for assembly of the extramembrane domains of mitochondrial β-barrel proteins (Figure 8). Certainly, the cytosol of a eukaryote presents a much more complex and protein-rich environment than the extracellular medium surrounding a bacterial cell, and factors that can assist substrate proteins into and out from the SAM complex would benefit the folding of β-barrels in the mitochondrial outer membrane.
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ACKNOWLEDGMENTS |
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for critical comments on the manuscript. This work was supported by grants from the Australian Research Council and National Health & Medical Research Council (to T.L.) and an Australian Postgraduate Award (to N.C.C.) |
Footnotes |
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Address correspondence to: Trevor Lithgow (t.lithgow{at}unimelb.edu.au)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Buchanan, S. K. (1999). Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr. Opin. Struct. Biol 9, 455–461.[CrossRef][Medline]
Daum, G., Bohni, P. C., and Schatz, G. (1982). Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem 257, 13028–13033.
Dolezal, P., Likic, V., Tachezy, J., and Lithgow, T. (2006). Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318.
Gabriel, K., Buchanan, S. K., and Lithgow, T. (2001). The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes. Trends Biochem. Sci 26, 36–40.[CrossRef][Medline]
Gabriel, K., Egan, B., and Lithgow, T. (2003). Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins. EMBO J 22, 2380–2386.[CrossRef][Medline]
Gentle, I., Gabriel, K., Beech, P., Waller, R., and Lithgow, T. (2004). The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol 164, 19–24.
Gentle, I. E., Burri, L., and Lithgow, T. (2005). Molecular architecture and function of the Omp85 family of proteins. Mol. Microbiol 58, 1216–1225.[Medline]
Gratzer, S., Lithgow, T., Bauer, R. E., Lamping, E., Paltauf, F., Kohlwein, S. D., Haucke, V., Junne, T., Schatz, G., and Horst, M. (1995). Mas37p, a novel receptor subunit for protein import into mitochondria. J. Cell Biol 129, 25–34.
Habib, S. J., Waizenegger, T., Niewienda, A., Paschen, S. A., Neupert, W., and Rapaport, D. (2007). The N-terminal domain of Tob55 has a receptor-like function in the biogenesis of mitochondrial beta-barrel proteins. J. Cell Biol 176, 77–88.
Humphries, A. D., Streimann, I. C., Stojanovski, D., Johnston, A. J., Yano, M., Hoogenraad, N. J., and Ryan, M. T. (2005). Dissection of the mitochondrial import and assembly pathway for human Tom40. J. Biol. Chem 280, 11535–11543.
Ishikawa, D., Yamamoto, H., Tamura, Y., Moritoh, K., and Endo, T. (2004). Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly. J. Cell Biol 166, 621–627.
Kamio, Y., and Nikaido, H. (1976). Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium. Biochemistry 15, 2561–2570.[CrossRef][Medline]
Kozjak, V., Wiedemann, N., Milenkovic, D., Lohaus, C., Meyer, H. E., Guiard, B., Meisinger, C., and Pfanner, N. (2003). An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J. Biol. Chem 278, 48520–48523.
Kozjak-Pavlovic, V., Ross, K., Benlasfer, N., Kimmig, S., Karlas, A., and Rudel, T. (2007). Conserved roles of Sam50 and metaxins in VDAC biogenesis. EMBO Rep 8, 576–582.[CrossRef][Medline]
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Meisinger, C. et al. (2007). The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria. EMBO J 26, 2229–2239.[CrossRef][Medline]
Meisinger, C. et al. (2004). The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane. Dev. Cell 7, 61–71.[CrossRef][Medline]
Meisinger, C., Wiedemann, N., Rissler, M., Strub, A., Milenkovic, D., Schonfisch, B., Muller, H., Kozjak, V., and Pfanner, N. (2006). Mitochondrial protein sorting: differentiation of beta-barrel assembly by Tom7-mediated segregation of Mdm10. J. Biol. Chem 281, 22819–22826.
Milenkovic, D., Kozjak, V., Wiedemann, N., Lohaus, C., Meyer, H. E., Guiard, B., Pfanner, N., and Meisinger, C. (2004). Sam35 of the mitochondrial protein sorting and assembly machinery is a peripheral outer membrane protein essential for cell viability. J. Biol. Chem 279, 22781–22785.
Model, K., Meisinger, C., Prinz, T., Wiedemann, N., Truscott, K. N., Pfanner, N., and Ryan, M. T. (2001). Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nat. Struct. Biol 8, 361–370.[CrossRef][Medline]
Nijtmans, L. G., Henderson, N. S., and Holt, I. J. (2002). Blue Native electrophoresis to study mitochondrial and other protein complexes. Methods 26, 327–334.[CrossRef][Medline]
Paschen, S. A., Neupert, W., and Rapaport, D. (2005). Biogenesis of beta-barrel membrane proteins of mitochondria. Trends Biochem. Sci 30, 575–582.[CrossRef][Medline]
Paschen, S. A., Waizenegger, T., Stan, T., Preuss, M., Cyrklaff, M., Hell, K., Rapaport, D., and Neupert, W. (2003). Evolutionary conservation of biogenesis of beta-barrel membrane proteins. Nature 426, 862–866.[CrossRef][Medline]
Pfanner, N., Wiedemann, N., Meisinger, C., and Lithgow, T. (2004). Assembling the mitochondrial outer membrane. Nat. Struct. Mol. Biol 11, 1044–1048.[CrossRef][Medline]
Ruiz, N., Falcone, B., Kahne, D., and Silhavy, T. J. (2005). Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121, 307–317.[CrossRef][Medline]
Sambrook, J., and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Habor, NY: Cold Spring Harbor Press.
Schleiff, E., and Soll, J. (2005). Membrane protein insertion: mixing eukaryotic and prokaryotic concepts. EMBO Rep 6, 1023–1027.[CrossRef][Medline]
Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.
Sogo, L. F., and Yaffe, M. P. (1994). Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane. J. Cell Biol 126, 1361–1373.
Voulhoux, R., Bos, M. P., Geurtsen, J., Mols, M., and Tommassen, J. (2003). Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299, 262–265.
Waizenegger, T., Habib, S. J., Lech, M., Mokranjac, D., Paschen, S. A., Hell, K., Neupert, W., and Rapaport, D. (2004). Tob38, a novel essential component in the biogenesis of beta-barrel proteins of mitochondria. EMBO Rep 5, 704–709.[CrossRef][Medline]
Waizenegger, T., Schmitt, S., Zivkovic, J., Neupert, W., and Rapaport, D. (2005). Mim1, a protein required for the assembly of the TOM complex of mitochondria. EMBO Rep 6, 57–62.[CrossRef][Medline]
Wiedemann, N., Kozjak, V., Chacinska, A., Schonfisch, B., Rospert, S., Ryan, M. T., Pfanner, N., and Meisinger, C. (2003). Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424, 565–571.[CrossRef][Medline]
Wittig, I., Braun, H. P., and Schagger, H. (2006). Blue native PAGE. Nat. Protoc 1, 418–428.[CrossRef][Medline]
Wu, T., Malinverni, J., Ruiz, N., Kim, S., Silhavy, T. J., and Kahne, D. (2005). Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121, 235–245.[CrossRef][Medline]
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