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The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria
Correspondence to Thomas Langer: Thomas.Langer{at}uni-koeln.de
Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.
Abbreviations used in this paper: CL, cardiolipin; PE, phosphatidylethanolamine; PS, phosphatidylserine; SGA, synthetic genetic array.
© 2009 Osman et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Introduction |
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Prohibitins form an evolutionary conserved family of membrane proteins with a variety of suggested activities in different cellular compartments (Rajalingam et al., 2005; Kasashima et al., 2006; Wang et al., 2008). Cell proliferation depends on prohibitins targeted to mitochondria (Merkwirth et al., 2008), where two homologous subunits, Phb1 and Phb2, assemble into multimeric, high–molecular weight complexes in the inner membrane (Coates et al., 1997; Berger and Yaffe, 1998; Tatsuta et al., 2005). Prohibitins have been found to be part of mitochondrial nucleoids (Bogenhagen et al., 2003), to affect the organization of mitochondrial DNA (Kasashima et al., 2008), and to protect endothelial cells against reactive oxygen–induced senescence (Kasashima et al., 2008; Schleicher et al., 2008). Fibroblasts lacking prohibitins show aberrant mitochondrial cristae and exhibit a decreased resistance toward apoptosis (Merkwirth et al., 2008). These deficiencies are caused by an impaired processing of the dynamin-like GTPase OPA1 (Merkwirth et al., 2008), a core component of the mitochondrial fusion machinery (Hoppins et al., 2007), which is mutated in dominant optic atrophy (Alexander et al., 2000; Delettre et al., 2000).
Despite recent progress in understanding of cellular roles of prohibitins, their molecular activity remained largely elusive (Mishra et al., 2006). Prohibitin complexes assemble with m-AAA proteases in the mitochondrial inner membrane and thereby modulate the turnover of nonnative membrane proteins (Steglich et al., 1999). Therefore, they have been proposed to exert chaperone-like activity (Nijtmans et al., 2002). However, the ring structure of purified prohibitin complexes (Tatsuta et al., 2005) and the sequence similarity of prohibitins to lipid raft–associated proteins of the SPFH family (Browman et al., 2007) are also consistent with a scaffolding function of prohibitin complexes in the inner membrane.
Notably, although they are required for embryonic development in mice, Caenorhabditis elegans, and Drosophila melanogaster, deletion of prohibitin genes in yeast leads to premature ageing but does not affect cell survival (Coates et al., 1997; Artal-Sanz et al., 2003; Merkwirth et al., 2008). We therefore searched for yeast genes whose function is essential for cell growth in the absence of prohibitins. Functional studies on the uncovered genetic network identified novel modulators of the phospholipid composition of the inner membrane and link the function of prohibitin complexes to cardiolipin (CL) and phosphatidylethanolamine (PE), nonbilayer phospholipids critical for mitochondrial structure and integrity.
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phb1 cells and a collection of 4,850 yeast strains lacking nonessential genes. Moreover, we tested candidate genes and reexamined previously described genetic interactors of prohibitins in the same yeast strain background. In this way, we identified 35 GEP genes (for genetic interactors of prohibitins), whose deletion caused lethality or severely impaired growth of prohibitin-deficient yeast cells on glucose-containing media (Table I). Consistent with a mitochondrial function of prohibitin complexes, 31 genes code for mitochondrial proteins. Interestingly, 19 of the genetic interactors fall into two functional classes: genes with functions during the assembly of the respiratory chain, and genes required for the maintenance of mitochondrial morphology (Dimmer et al., 2002) and the assembly of β-barrel proteins in the outer membrane (Meisinger et al., 2007). The latter group includes the genes MMM1, MDM10, MDM12, MDM31, MDM32, and MDM34, which were previously found to genetically interact with each other (Dimmer et al., 2005). Diverse functions have been associated with another group of eight GEP genes, which include the synthesis of CL (by the CL synthase Crd1) and the synthesis of PE (by the phosphatidylserine [PS] decarboxylase Psd1). Finally, growth of prohibitin-deficient cells was found to depend on eight GEP genes of unknown function (Table I).
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gep1phb1 cells expressing Phb1 from a tetracycline-regulatable promoter ([PHB1]). These cells grew normally on both fermentable and nonfermentable carbon sources under nonrepressing conditions, whereas addition of the tetracycline analogue doxycycline shut off PHB1 expression and inhibited cell growth (Fig. 1 B). Immunoblot analysis of cell extracts revealed that outer membrane proteins accumulated normally in these cells (Fig. 1 C). However, the loss of Phb1 was accompanied by a decrease of mitochondrial inner membrane and matrix proteins, which suggests functional deficits in the inner membrane (Fig. 1 C). We therefore determined in Phb1-depleted cells the formation of the mitochondrial membrane potential, which is required for protein transport across and into the inner membrane and thereby essential for cell survival. Depletion of Phb1 decreased the membrane potential in phb1[PHB1] cells and led to its complete dissipation in gep1phb1[PHB1] cells (Fig. 1 D). These findings point to essential functions of both genes for the integrity of the inner membrane and provide an explanation for the synthetic lethal interaction of GEP1 and PHB1.
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gep1, or phb1 cells and gep1 or gep1phb1 cells harboring [PHB1]. An inspection of these cells by fluorescence microscopy revealed the accumulation of ball-shaped and clustered mitochondria in gep1phb1 cells upon down-regulation of Phb1 (Fig. 2 A). In the absence of doxycycline, mitochondrial morphology was slightly impaired in these cells, most likely reflecting a deleterious effect of Phb1 overexpression. Notably, the aberrant morphology of mitochondria was not a consequence of cell death, as mitochondria were inspected while at least 85% of the cells were still viable as determined by FUN-1 staining (unpublished data). Deletion of PHB1 alone did not interfere with the formation of tubular mitochondria (Fig. 2 A).
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40% of gep1phb1[PHB1] but not phb1[PHB1] cells depleted of Phb1 (Fig. 2 B). We carefully quantified the surface of cristae membranes and related it to the surface of the mitochondrial contours in ultrathin sections of Gep1- and Phb1-deficient cells (Fig. 2 C). The surface of cristae membranes decreased only slightly in phb1[PHB1] cells but was reduced dramatically by 80% in gep1phb1[PHB1] cells upon down-regulation of Phb1 (Fig. 2 C), which demonstrates that Gep1 and Phb1 concomitantly affect mitochondrial cristae morphogenesis.
When cells were grown on glycerol-containing medium, 80% of gep1 and 60% of phb1 cells contained an at least partially fragmented mitochondrial network, which indicates that both proteins are crucial for mitochondrial morphology under conditions of increased mitochondrial demand (Fig. 3 A). Inner membrane fusion and the formation of cristae depend on the dynamin-like GTPase Mgm1 (Meeusen et al., 2006), which undergoes proteolytic processing by the rhomboid protease Pcp1 in the inner membrane. Cleavage results in the accumulation of two isoforms, L- and S-Mgm1, that functionally cooperate during inner membrane fusion (Sesaki et al., 2003). Interestingly, deletion of GEP1 significantly impaired the formation of S-Mgm1 in cells grown under respiring conditions (Fig. 3 B). These findings identify Gep1 as a novel modulator of Mgm1 processing in the inner membrane and suggest that the unbalanced accumulation of L- and S-Mgm1 causes deficiencies in cristae morphogenesis in gep1 mitochondria.
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gep1phb1 cells expressing plasmid-borne PHB1 were transformed with a multicopy yeast library. After plasmid shuffling, the previously uncharacterized GEP2 gene (YDR185c), which codes for a homologue of Gep1, was identified as a multicopy suppressor of the synthetic lethal interaction of gep1 and phb1 (Fig. 4 A), indicating overlapping functions of members of this conserved protein family.
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gep1phb1 cells, linking functional defects in these cells to the cellular phospholipid metabolism (Fig. 4 A). PS, which is synthesized by Cho1 in the ER, is transported to mitochondria and decarboxylated to form PE by Psd1 in the inner membrane (Voelker, 2005). Psd1 is a bifunctional protein, which is required for both PE synthesis and the regulation of multidrug resistance (Gulshan et al., 2008). It has been observed to genetically interact with prohibitins (Table I; Birner et al., 2003), indicating a complex network of genetic interactions between prohibitins, Gep1, and the cellular phospholipid metabolism.
We therefore determined the mitochondrial phospholipid profile in gep1 cells by TLC. Whereas the majority of mitochondrial phospholipids accumulated at normal levels in the absence of Gep1, PE was present at significantly reduced levels in gep1 mitochondria (Fig. 4 B). Overexpression of Cho1 or Gep2, but not of Ups1, restored normal PE levels in Gep1-deficient mitochondria (Fig. 4 B). Further genetic experiments corroborated the role of Gep1 for the formation of cellular PE. Cell survival depends on PE, which is synthesized either by Psd1 within mitochondria or by Psd2 in the Golgi apparatus (Voelker, 2004). To examine which of these pathways is affected by Gep1, we deleted GEP1 in psd1 and psd2 cells and examined cell growth both on fermentable and nonfermentable carbon sources (Fig. 4 C). The absence of Gep1 did not impair growth of cells deficient of Psd1 at any temperature, which is consistent with an epistatic relationship of these genes (Fig. 4 C). However, growth of gep1psd2 cells was inhibited on nonfermentable carbon sources at 37°C (Fig. 4 C), which suggests that Gep1 specifically affects mitochondrial PE. The synthesis of PE within mitochondria was previously found to be essential for the viability of yeast mutants lacking the CL synthase Crd1, which is localized in the mitochondrial inner membrane (Gohil et al., 2005). Consistent with the significantly reduced levels of PE in gep1 mitochondria, we did not obtain viable double mutant offspring after tetrad dissection of diploid cells heterozygous for deletions in GEP1 and CRD1 indicating synthetic lethality (Fig. 4 D). We conclude from these experiments that Gep1 is required for the accumulation of PE in mitochondria.
These findings raise the possibility that an altered phospholipid composition of the inner membrane impairs Mgm1 cleavage and causes an altered cristae morphogenesis in the absence of Gep1. We therefore assessed processing of Mgm1 in mitochondria lacking Psd1 or Crd1 (Fig. 3 B). Deletion of PSD1, as well as that of GEP1, impaired the formation of S-Mgm1, whereas Mgm1 cleavage was not affected in the absence of CRD1 under these conditions (Fig. 3 B). We conclude that the phospholipid composition, in particular the PE content, is crucial for efficient Mgm1 processing by rhomboid in the inner membrane.
Gep1 is dispensable for PE synthesis but controls its stability in mitochondria
To unravel the molecular basis of impaired PE accumulation in the absence of Gep1, we first determined the localization of Gep1 within mitochondria. A Gep1 variant carrying a C-terminal hemagglutinin tag was expressed in gep1 cells and in gep1phb1 cells. Expression of this variant promoted growth of gep1phb1 cells, demonstrating that the C-terminal extension did not interfere with Gep1 function (unpublished data). Mitochondria were isolated from gep1 cells and subjected to protease treatment. Gep1 was degraded only under hypotonic conditions, which resulted in osmotic disruption of the outer membrane, indicating that Gep1 is localized in the intermembrane space (Fig. 5 A). A hydropathy blot did not provide any evidence for the presence of a membrane-spanning segment in Gep1. However, Gep1 was found in the pellet fraction after carbonate extraction of mitochondrial membranes, which indicates a tight membrane association (Fig. 5 A).
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gep1 mitochondria. After osmotic disruption of the outer membrane, mitoplasts were incubated with fluorescently labeled PS (NBD-PS), which is converted to NBD-PE (Fig. 5 B). NBD-PE did not accumulate in psd1 mitochondria, which demonstrates that NBD-PE is formed by Psd1. The synthesis of NBD-PE was not affected in the absence of Gep1 (Fig. 5 B). We observed a slightly but significantly increased rate of NBD-PE synthesis in gep1 mitochondria, which may indicate an alleviated product inhibition of Psd1 in these mitochondria.
These results were substantiated by pulse-labeling experiments in vivo, which were performed in a psd2 strain background to exclude masking effects of nonmitochondrial PE synthesis (Fig. 5 C). We labeled psd2 and psd2gep1 cells with [3H]serine and monitored the incorporation of 3H in PE within mitochondria. Deletion of GEP1 did not affect mitochondrial PE synthesis by mitochondrial Psd1 (Fig. 5 C). Pulse chase experiments, however, revealed a significantly decreased stability of newly synthesized PE in the absence of Gep1 without a significant accumulation of other lipid species (Fig. 5 D). We conclude that the decreased PE concentration in the absence of Gep1 is not caused by an impaired Psd1 activity or uptake of its substrate PS but instead reflects a reduced stability of PE within gep1 mitochondria.
Lipid-specific functions of Gep1-like proteins for PE and CL
Several lines of evidence point to overlapping activities of Gep1 family members: first, both gep1 and ups1 show a synthetic lethal interaction with prohibitin mutants (Table I); and second, overexpression of Gep2 promoted growth of gep1phb1 cells and restored PE accumulation in Gep1-deficient mitochondria (Fig. 4 A, B). We therefore determined the accumulation of PE in gep1, gep2, and ups1 mitochondria both by TLC and by mass spectrometry (Fig. 6, A and B). In contrast to GEP1, deletion of GEP2 and UPS1 did not affect PE levels within mitochondria (Fig. 6, A and B). Our analysis revealed, however, a crucial role of Ups1 for CL levels. CL is a dimeric phosphoglycerolipid predominantly present in mitochondria, where it is synthesized by the CL synthase Crd1 in the inner membrane (Schlame, 2008). CL was decreased approximately sevenfold in ups1 mitochondria but remained unaffected in the absence of Gep1 or Gep2 (Fig. 6, A and B). Growth of ups1 cells was severely impaired on glucose-containing medium (Fig. 6 C), a phenotype reminiscent of yeast cells lacking Pgs1 that catalyzes the rate-limiting step of CL biosynthesis. Deletion of GEP1 and GEP2, however, did not affect cell growth under these conditions (Fig. 6 C). Similar to cells lacking Psd1, gep1 cells exhibited an increased tendency to lose mitochondrial DNA under these conditions (unpublished data). We conclude that the accumulation of CL in mitochondria depends on Ups1, whereas Gep1 controls mitochondrial PE.
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ups1 mitochondria (Fig. 6, A and B) and suppressed growth defects associated with the loss of Ups1 (Fig. 6 C). In contrast, neither CL levels nor cell growth were affected upon deletion of GEP2 in ups1 cells, although Gep1 and Gep2 share 55% identical amino acids (Fig. 6, A–C). PE, however,remained reduced in gep1ups1 mitochondria and accumulated at similar levels as in gep1 mitochondria (Fig. 6, A and B), which demonstrates that the absence of Ups1 does not alleviate the requirement of Gep1 for the accumulation of PE. Thus, a complex functional network of Gep1-like proteins controls mitochondrial PE and CL. To further define the functional interdependence of PE and CL pathways, we generated yeast strains overexpressing Gep1, Gep2, or Ups1 from galactose-inducible promoters. Overexpression of Gep1 impaired cell growth on galactose-containing medium, whereas increased cellular levels of Gep2 or Ups1 did not interfere with cell growth (Fig. 6 F). Mitochondria were isolated from these cells, and the phospholipid profile was determined by TLC and mass spectrometry (Fig. 6, D and E). In agreement with the observed growth defect, we noted a significantly reduced CL content of mitochondria containing overexpressed Gep1 (Fig. 6 D, E). Moreover, PE levels were increased, whereas other phospholipids were present at normal levels in these mitochondria (Fig. 6, D and E; and unpublished data). PE and CL were not altered in a statistically significant manner upon overexpression of Ups1 or Gep2 (Fig. 6, D and E).
Collectively, these experiments define lipid-specific activities of Gep1-like proteins for CL and PE and, at the same time, point to common steps in the regulation of both phospholipids within mitochondria. Both the observed restoration of CL levels in ups1 cells upon deletion of GEP1 and the reduction of CL levels upon Gep1 overexpression suggest a competition between Gep1 and Ups1.
Survival of prohibitin-deficient cells depends on the lipid composition of mitochondrial membranes
The regulation of the phospholipid composition of the inner membrane by Gep1 and Ups1 proteins together with their synthetic lethal interaction with prohibitins suggests that reduced levels of PE and CL are deleterious for inner membrane integrity in prohibitin-deficient cells. Accordingly, other GEP genes may affect the PE and CL levels in mitochondrial membranes as well. We therefore isolated mitochondria from various yeast strains lacking Phb1 or GEP genes, extracted membrane lipids, and determined PE and CL levels by mass spectrometry (Fig. 7). Strikingly, mitochondrial PE and/or CL were affected in the majority of 23 examined strains (Fig. 7). Only some strains showed normal PE and almost unaltered CL levels in mitochondria (Fig. 7). The latter group included cells lacking assembly factors of the FO particle of the F1FO ATP-synthase, like Atp10 and Atp23, that have been previously found to interact genetically with prohibitins (Osman et al., 2007), or Oxa1, for which a function during FO assembly was recently described (Jia et al., 2007). Deletion of PSD1 or CRD1 resulted in the expected drastic reduction of the PE or CL content of mitochondrial membranes, respectively (Fig. 7). Notably, we observed increased PE levels in cells showing a severely reduced CL content. This is in agreement with previous findings describing an increase in mitochondrial PE in crd1 cells, and suggested a coordinated regulation of both phospholipids (Zhong et al., 2004). Moreover, membranes isolated from Phb1-deficient mitochondria contained reduced amounts of CL and slightly increased PE levels. Strikingly, the loss of a large number of GEP genes genetically interacting with prohibitins led to strongly reduced levels of PE and/or CL (Fig. 7). These included genes with functions for mitochondrial morphology and the assembly of β-barrel proteins (MDM10, MMM1, MDM31, MDM32, MDM34, and MDM35; Merz et al., 2007; Bolender et al., 2008), genes associated with the assembly of respiratory chain complexes (COX6, YTA10, and YTA12), and several uncharacterized open reading frames (GEP3-6). Our findings link the function of these genes to the mitochondrial lipid metabolism and point to a critical role of the PE and CL content of the inner membrane for the survival of prohibitin-deficient cells.
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ups1 cells restored CL levels in mitochondria, which suggests competition between Gep1 and Ups1. Consistently, overexpression of Gep1 reduces CL in mitochondria. Thus, the phospholipid content of mitochondrial membranes critically depends on the level of Gep1. The competitive action of Gep1 and Ups1 allows the adjustment of relative PE and CL levels simply by modulating the amounts or the availability of the regulatory proteins. These findings demonstrate that mitochondrial levels of PE and CL are regulated coordinately by related proteins and are therefore in agreement with previous notions that cells may require a critical amount of these nonbilayer-forming phospholipids (Zhong et al., 2004; Gohil et al., 2005). Gep1-like proteins likely exert a regulatory role during membrane biogenesis, as the mitochondrial phospholipid profile is only modestly altered in cells lacking all Gep1-like proteins. The decrease of the PE content of mitochondrial membranes in the absence of Gep1 is not caused by an impaired PE synthesis. Rather, PE does not accumulate stably in Gep1-deficient mitochondria, suggesting that Gep1 inhibits either a PE-specific lipase or the export of PE from mitochondria. Accordingly, the competition of Gep1 and Ups1 may determine the specificity of mitochondrial phospholipases or lipid transport processes. In agreement with an inhibitory role of Gep1 for PE export from mitochondria, we observed in the absence of Gep1 a slight increase of PC, which is generated by methyl transferases in ER membranes using mitochondrial PE (Kodaki and Yamashita, 1989). Contact sites between inner and outer mitochondrial membranes have been discussed as sites of phospholipid transport (Simbeni et al., 1990). It will be therefore of interest to examine whether Gep1-like proteins locate to these sites.
We identify the morphogenesis of cristae as one process critically dependent on mitochondrial PE. Decreased PE levels in Gep1- or Psd1-deficient cells compromise the processing of the dynamin-like GTPase Mgm1, which is required for membrane fusion and cristae formation (Meeusen et al., 2006). Under these conditions, prohibitins are essential to maintain inner membrane integrity. Thus, residual Mgm1 processing sufficient to maintain mitochondrial cristae at decreased PE levels appears to critically depend on prohibitins. Notably, we have recently identified the processing of the mammalian Mgm1 homologue OPA1 as the central process controlled by prohibitins in mouse fibroblasts (Merkwirth et al., 2008), which indicates that the same processes depend on prohibitin function in evolutionary distant organisms. Therefore, phenotypic differences associated with the loss of prohibitins in yeast and mammals likely reflect differences in the phospholipid profile of mitochondrial membranes or the lipid dependence of Mgm1/OPA1 processing itself.
Although the absence of CL did not inhibit Mgm1 processing in our experiments, we do not exclude a role of CL for proteolytic cleavage under certain growth conditions in yeast or in other organisms. Variations of the PE content of the inner membrane may mask the dependence of Mgm1 processing on CL. Accordingly, differences in the relative content of PE and CL may explain why the loss of Ups1 was observed previously to inhibit Mgm1 processing (Sesaki et al., 2006). It is therefore an intriguing possibility that impaired processing of the mammalian Mgm1 homologue OPA1 causes the disturbed formation of mitochondrial cristae, which was observed in lymphoblasts of Barth syndrome patients or yeast cells lacking the CL transacylase tafazzin (Acehan et al., 2007; Claypool et al., 2008). The identification of Gep1-like proteins as regulators of CL and PE now allows for the direct examination of the role of an altered phospholipid composition for mitochondrial dysfunction in disease.
Gep1-like proteins and prohibitins acting as membrane organizers may affect various membrane-associated processes, which are known to depend on nonbilayer phospholipids. Mitochondrial fusion requires phospholipase D, which hydrolyzes CL in the outer membrane and generates the fusogenic lipid phosphatidic acid (Choi et al., 2006). CL and PE affect also the insertion and oligomerization of the proapoptotic Bcl2-family member Bax in the outer membrane (Lucken-Ardjomande et al., 2008). A CL deficiency in the inner membrane impairs the activity of mitochondrial enzymes (Jiang et al., 2000), decreases the stability of respiratory chain supercomplexes and of mitochondrial DNA (Pfeiffer et al., 2003), and accelerates apoptosis by facilitating the release of cytochrome c from the intramitochondrial storage compartments (Choi et al., 2007). It is conceivable that the assembly of the F1FO ATP-synthase and respiratory complexes is yet another process dependent on defined functional domains within mitochondrial membranes. This is suggested by the synthetic interaction of prohibitins with genes coding for assembly factors of inner membrane complexes (Osman et al., 2007), which do not drastically affect mitochondrial PE or CL levels when deleted.
Interestingly, mutations in the majority of GEP genes, which show a synthetic interaction with prohibitins, cause decreased levels of nonbilayer lipids. This includes MDM10 and MMM1, which were originally identified as genes required for mitochondrial inheritance and DNA stability (Boldogh et al., 1998) but were found more recently to function in the assembly of β-barrel proteins in the outer membrane (Meisinger et al., 2007). Accordingly, an impaired insertion of an outer membrane protein affecting mitochondrial lipid biosynthesis may explain the reduced levels of PE and CL in the absence of Mdm10 or Mmm1. However, it cannot be excluded that both proteins play a direct role for the mitochondrial lipid metabolism and that an altered lipid composition causes deficiencies in the assembly of outer membrane proteins. Similarly, it is an attractive possibility that at least some of the GEP genes identified in this study, including previously uncharacterized open reading frames, regulate directly the biosynthesis of PE and CL in mitochondria and affect the activity of biosynthetic enzymes or intramitochondrial lipid transport processes. The identification of components mediating lipid import from the ER, transbilayer flipping across mitochondrial membranes, or lipid transport across the intermembrane space of mitochondria has yet be accomplished.
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phb1 cells was performed as described previously (Tong et al., 2001). Synthetic genetic interactions with phb1 were confirmed by sporulation and tetrad dissection. In agreement with the functional interdependence of Phb1 and Phb2, the same interactors were identified in SGAs using phb2 cells. To identify multicopy suppressors of synthetic lethal interactions, phb1gep1[PHB1] cells were transformed with a genomic Yep13 high-copy library. After growth for 1 d at 30°C on SC-Leu, plates were replicated on plates containing 5' fluoroorotic acid (1 mg/ml). Clones were analyzed after 2 d, and the suppressor gene was identified by subcloning. The genotypes of yeast strains used are described in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200810189/DC1).
Microscopy
Fluorescence and electron microscopy was performed as described previously (Dürr et al., 2006) using a microscope (Axioplan 2; Carl Zeiss, Inc.) equipped with a Plan-Neofluar 100x/1.30 NA Ph3 oil objective lens (Carl Zeiss, Inc.). Images were recorded with a monochrome camera (Evolution VF Mono Cooled; Intas) and processed with Image-Pro Plus 5.0 and Scope Pro4.5 software (Media Cybernetics). For the determination of the cristae/mitochondrial contour ratio, ultrathin sections were viewed in a transmission electron microscope (JEM-2100; JEOL Ltd.) and images were taken with a digital camera (Erlangshen ES500W, model 782; Gatan Inc.). Approximately 100 micrographs of mitochondria were taken for each strain at the same magnification. For each image, the numbers of cristae were counted and the contour of each mitochondrion was measured in micrometers by using the measure function of ImageJ. For each strain the numbers of cristae were put into relation to the sum of the mitochondrial contours. Processing of Mgm1 was monitored by immunoblotting using Mgm1-specific antiserum, provided by A. Reichert (University of Frankfurt, Frankfurt, Germany).
Lipid analysis
TLC analysis.
Mitochondria were isolated from cells grown on yeast-peptone (YP) medium containing 2% galactose and 0.5% lactate. The mitochondrial fraction was washed, resuspended in buffer A (0.6 M sorbitol and 5 mM MES, pH 6), and loaded on a continuous sucrose gradient (20–50% in buffer A). Mitochondria were harvested from the lower third of the gradient and diluted 1:5 in buffer A, pelleted, washed once in SEM buffer (250 mM sucrose, 10 mM MOPS, pH 7.2, and 1 mM EDTA), and finally resuspended in SEM buffer. The purity of the mitochondrial fraction and absence of contaminations by vacuolar and ER membranes was assessed by immunoblotting using Sec61- and Vac8-specific antisera, provided by T. Sommer (Max-Delbrück-Centre, Berlin, Germany) and C. Ungermann (University of Osnabrück, Osnabrück, Germany), respectively. Purified mitochondria (1 mg) were mixed with 1.5 ml chloroform/methanol (1:1, vol/vol) and vigorously shaken for 60 min. 300 µl H2O was added and samples were vortexed for 60 s. After centrifugation (1,000 rpm, 5 min), the aqueous phase was removed and the solvent phase was washed with 250 µl methanol/H2O (1:1, vol/vol). Samples were then dried under a constant stream of air. Lipids were dissolved in chloroform, phosphate concentration was determined (Rouser et al., 1970), and samples were subjected to TLC analysis. TLC plates (HPTLC; Merck & Co., Inc.) were developed with chloroform/methanol/25% ammonia (50:50:3, vol/vol/vol) when not stated otherwise, allowing the separation of PC, PE, and CL from other mitochondrial phospholipids (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200810189/DC1). TLC plates were stained with 470 mM CuSO4 in 8.5% o-phosphoric acid and subsequently incubated for 10 min at 180°C.
Quantification of PE and CL by mass spectrometry.
Mitochondrial lipids were extracted and phosphate concentration was determined according to Rouser et al. (1970). For mass spectrometric analysis, 1.5-nmol phospholipids of mitochondrial fractions were extracted in the presence of CL (CL56:0; Avanti Polar Lipids, Inc.) and PE standard (50 pmol each) as described previously (Brügger et al., 2006). Dried lipids were redissolved in 10 mM ammonium acetate in methanol.
Quantification of PE was performed by neutral-loss scanning, selecting for a neutral loss of 141 D as described previously (Brügger et al., 2006). Quantification of CL was performed in negative ion mode on a quadrupole time-of-flight mass spectrometer (QStar Elite, Applied Biosystems). 10 µl of lipid extracts was diluted 1:2 with 0.1% piperidine in methanol and automatically infused (Triversa Nanomate; Advion Biosciences). The ionization voltage was set to –0.95 kV and gas pressure was set to 0.5 psi. CLs were detected as single charged molecules. CL species (all combinations of fatty acids 16:0, 16:1, 16:2, 18:0, 18:1, and 18:2) were analyzed by targeted product ion scanning. The peak areas of CL-derived fatty acid fragments were extracted from the respective product ion spectra via the "Extract Fragments" script (Analyst QS 2.0; Applied Biosystems). Isotope correction for M+2 ions was performed manually, and values were corrected for response factors of standards.
Determination of Psd1 activity.
Mitochondria were resuspended in assay buffer B (0.1 M Tris/HCl, pH 7.4, 10 mM EDTA, and 2 µM PS-C6-NBD [Avanti Polar Lipids]) to a final concentration of 5 mg/ml and incubated at 25°C. At the time points indicated, mitochondria (500 µg) were removed from the reaction mixture, and phospholipids were extracted and analyzed by TLC (developing solvent: chloroform/methanol/H2O/triethylamine; 30:35:7:35 vol/vol/vol/vol). NBD signals were detected and quantified by fluor imaging (TyphoonTrio; GE Healthcare).
In vivo labeling of phospholipids.
Logarithmically growing yeast cells were harvested and resuspended in medium supplemented with [3H]serine (12.5 µCi/ml) to a final OD600 of 5. After incubation at 30°C for 0, 20, 40, or 60 min, cells corresponding to 10 OD600 were harvested and stored in liquid nitrogen. Phospholipids were extracted and analyzed by TLC. PE spots were recovered from the TLC plates and mixed with 400 µl H2O and 8 ml of scintillation cocktail. Labeled PE was quantified by liquid scintillation counting. The stability of PE was monitored in pulse chase experiments. Yeast cells were labeled for 10 min as described above. Cells were then harvested, resuspended in medium containing 20 mM of unlabeled serine, and incubated at 30°C for 0, 30, 60, 90, 120, or 150 min before PE was quantified.
Online supplemental material
Fig. S1 documents the resolution of the TLC system applied in this study. Table S1 lists yeast strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200810189/DC1.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft to T. Langer (SFB635/C4) and B. Westermann (WE2174/4-1), and support from the German-Israeli Project (DIP grant F.5.1) and the European Research Counsel to T. Langer.
Submitted: 31 October 2008
Accepted: 13 January 2009
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References |
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Acehan, D., Y. Xu, D.L. Stokes, and M. Schlame. 2007. Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography. Lab. Invest. 87:40–48.[CrossRef][Medline]
Alexander, C., M. Votruba, U.E. Pesch, D.L. Thiselton, S. Mayer, A. Moore, M. Rodriguez, U. Kellner, B. Leo-Kottler, G. Auburger, et al. 2000. OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat. Genet. 26:211–215.[CrossRef][Medline]
Artal-Sanz, M., W.Y. Tsang, E.M. Willems, L.A. Grivell, B.D. Lemire, H. van der Spek, L.G. Nijtmans, and M.A. Sanz. 2003. The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans. J. Biol. Chem. 278:32091–32099.
Berger, K.H., and M.P. Yaffe. 1998. Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae. Mol. Cell. Biol. 18:4043–4052.
Birner, R., R. Nebauer, R. Schneiter, and G. Daum. 2003. Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae. Mol. Biol. Cell. 14:370–383.
Bogenhagen, D.F., Y. Wang, E.L. Shen, and R. Kobayashi. 2003. Protein components of mitochondrial DNA nucleoids in higher eukaryotes. Mol. Cell. Proteomics. 2:1205–1216.
Boldogh, I., N. Vojtov, S. Karmon, and L.A. Pon. 1998. Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p. J. Cell Biol. 141:1371–1381.
Bolender, N., A. Sickmann, R. Wagner, C. Meisinger, and N. Pfanner. 2008. Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep. 9:42–49.[CrossRef][Medline]
Browman, D.T., M.B. Hoegg, and S.M. Robbins. 2007. The SPFH domain-containing proteins: more than lipid raft markers. Trends Cell Biol. 17:394–402.[CrossRef][Medline]
Brügger, B., B. Glass, P. Haberkant, I. Leibrecht, F.T. Wieland, and H.G. Krausslich. 2006. The HIV lipidome: a raft with an unusual composition. Proc. Natl. Acad. Sci. USA. 103:2641–2646.
Chan, D.C. 2006. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 125:1241–1252.[CrossRef][Medline]
Choi, S.Y., P. Huang, G.M. Jenkins, D.C. Chan, J. Schiller, and M.A. Frohman. 2006. A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis. Nat. Cell Biol. 8:1255–1262.[CrossRef][Medline]
Choi, S.Y., F. Gonzalvez, G.M. Jenkins, C. Slomianny, D. Chretien, D. Arnoult, P.X. Petit, and M.A. Frohman. 2007. Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis. Cell Death Differ. 14:597–606.[CrossRef][Medline]
Claypool, S.M., P. Boontheung, J.M. McCaffery, J.A. Loo, and C.M. Koehler. 2008. The cardiolipin transacylase, tafazzin, associates with two distinct respiratory components providing insight into Barth syndrome. Mol. Biol. Cell. 19:5143–5155.
Coates, P.J., D.J. Jamieson, K. Smart, A.R. Prescott, and P.A. Hall. 1997. The prohibitin family of mitochondrial proteins regulate replicative lifespan. Curr. Biol. 7:607–610.[CrossRef][Medline]
de Kruijff, B. 1997. Lipid polymorphism and biomembrane function. Curr. Opin. Chem. Biol. 1:564–569.[CrossRef][Medline]
Dee, C.T., and K.G. Moffat. 2005. A novel family of mitochondrial proteins is represented by the Drosophila genes slmo, preli-like and real-time. Dev. Genes Evol. 215:248–254.[CrossRef][Medline]
Delettre, C., G. Lenaers, J.M. Griffoin, N. Gigarel, C. Lorenzo, P. Belenguer, L. Pelloquin, J. Grosgeorge, C. Turc-Carel, E. Perret, et al. 2000. Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genet. 26:207–210.[CrossRef][Medline]
Dimmer, K.S., S. Fritz, F. Fuchs, M. Messerschmitt, N. Weinbach, W. Neupert, and B. Westermann. 2002. Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae. Mol. Biol. Cell. 13:847–853.
Dimmer, K.S., S. Jakobs, F. Vogel, K. Altmann, and B. Westermann. 2005. Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast. J. Cell Biol. 168:103–115.
Dürr, M., M. Escobar-Henriques, S. Merz, S. Geimer, T. Langer, and B. Westermann. 2006. Nonredundant roles of mitochondria-associated F-box proteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology in yeast. Mol. Biol. Cell. 17:3745–3755.
Frick, M., N.A. Bright, K. Riento, A. Bray, C. Merrified, and B.J. Nichols. 2007. Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding. Curr. Biol. 17:1151–1156.[CrossRef][Medline]
Gohil, V.M., M.N. Thompson, and M.L. Greenberg. 2005. Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine and cardiolipin biosynthetic pathways in Saccharomyces cerevisiae. J. Biol. Chem. 280:35410–35416.
Gulshan, K., J.A. Schmidt, P. Shahi, and W.S. Moye-Rowley. 2008. Evidence for the bifunctional nature of mitochondrial phosphatidylserine decarboxylase: role in Pdr3-dependent retrograde regulation of PDR5 expression. Mol. Cell. Biol. 28:5851–5864.
Hoppins, S., L. Lackner, and J. Nunnari. 2007. The machines that divide and fuse mitochondria. Annu. Rev. Biochem. 76:751–780.[CrossRef][Medline]
Janitor, M., M. Obernauerova, S.D. Kohlwein, and J. Subik. 1996. The pel1 mutant of Saccharomyces cerevisiae is deficient in cardiolipin and does not survive the disruption of the CHO1 gene encoding phosphatidylserine synthase. FEMS Microbiol. Lett. 140:43–47.[Medline]
Jia, L., M.K. Dienhart, and R.A. Stuart. 2007. Oxa1 directly interacts with Atp9 and mediates its assembly into the mitochondrial F1Fo-ATP synthase complex. Mol. Biol. Cell. 18:1897–1908.
Jiang, F., M.T. Ryan, M. Schlame, M. Zhao, Z. Gu, M. Klingenberg, N. Pfanner, and M.L. Greenberg. 2000. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function. J. Biol. Chem. 275:22387–22394.
Kasashima, K., E. Ohta, Y. Kagawa, and H. Endo. 2006. Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. J. Biol. Chem. 281:36401–36410.
Kasashima, K., M. Sumitani, M. Satoh, and H. Endo. 2008. Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids. Exp. Cell Res. 314:988–996.[CrossRef][Medline]
Kodaki, T., and S. Yamashita. 1989. Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption. Eur. J. Biochem. 185:243–251.[Medline]
Kumar, A., S. Agarwal, J.A. Heyman, S. Matson, M. Heidtman, S. Piccirillo, L. Umansky, A. Drawid, R. Jansen, Y. Liu, et al. 2002. Subcellular localization of the yeast proteome. Genes Dev. 16:707–719.
Langhorst, M.F., A. Reuter, F.A. Jaeger, F.M. Wippich, G. Luxenhofer, H. Plattner, and C.A. Stuermer. 2008. Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2. Eur. J. Cell Biol. 87:211–226.[CrossRef][Medline]
Lucken-Ardjomande, S., S. Montessuit, and J.C. Martinou. 2008. Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane. Cell Death Differ. 15:929–937.[CrossRef][Medline]
Matsumoto, K., J. Kusaka, A. Nishibori, and H. Hara. 2006. Lipid domains in bacterial membranes. Mol. Microbiol. 61:1110–1117.[CrossRef][Medline]
McBride, H.M., M. Neuspiel, and S. Wasiak. 2006. Mitochondria: more than just a powerhouse. Curr. Biol. 16:R551–R560.[CrossRef][Medline]
Meeusen, S., R. DeVay, J. Block, A. Cassidy-Stone, S. Wayson, J.M. McCaffery, and J. Nunnari. 2006. Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell. 127:383–395.[CrossRef][Medline]
Meisinger, C., S. Pfannschmidt, M. Rissler, D. Milenkovic, T. Becker, D. Stojanovski, M.J. Youngman, R.E. Jensen, A. Chacinska, B. Guiard, 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]
Merkwirth, C., S. Dargazanli, T. Tatsuta, S. Geimer, B. Lower, F.T. Wunderlich, J.C. von Kleist-Retzow, A. Waisman, B. Westermann, and T. Langer. 2008. Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria. Genes Dev. 22:476–488.
Merz, S., M. Hammermeister, K. Altmann, M. Dürr, and B. Westermann. 2007. Molecular machinery of mitochondrial dynamics in yeast. Biol. Chem. 388:917–926.[CrossRef][Medline]
Mishra, S., L.C. Murphy, and L.J. Murphy. 2006. The prohibitins: emerging roles in diverse functions. J. Cell. Mol. Med. 10:353–363.[Medline]
Nebauer, R., I. Schuiki, B. Kulterer, Z. Trajanoski, and G. Daum. 2007. The phosphatidylethanolamine level of yeast mitochondria is affected by the mitochondrial components Oxa1p and Yme1p. FEBS J. 274:6180–6190.[CrossRef][Medline]
Nijtmans, L.G., S.M. Artal, L.A. Grivell, and P.J. Coates. 2002. The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease. Cell. Mol. Life Sci. 59:143–155.[CrossRef][Medline]
Osman, C., C. Wilmes, T. Tatsuta, and T. Langer. 2007. Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase. Mol. Biol. Cell. 18:627–635.
Pfeiffer, K., V. Gohil, R.A. Stuart, C. Hunte, U. Brandt, M.L. Greenberg, and H. Schagger. 2003. Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol. Chem. 278:52873–52880.
Rajalingam, K., C. Wunder, V. Brinkmann, Y. Churin, M. Hekman, C. Sievers, U.R. Rapp, and T. Rudel. 2005. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat. Cell Biol. 7:837–843.[CrossRef][Medline]
Reichert, A.S., and W. Neupert. 2002. Contact sites between the outer and inner membrane of mitochondria-role in protein transport. Biochim. Biophys. Acta. 1592:41–49.[Medline]
Rouser, G., S. Fkeischer, and A. Yamamoto. 1970. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 5:494–496.[Medline]
Schlame, M. 2008. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes. J. Lipid Res. 49:1607–1620.
Schleicher, M., B.R. Shepherd, Y. Suarez, C. Fernandez-Hernando, J. Yu, Y. Pan, L.M. Acevedo, G.S. Shadel, and W.C. Sessa. 2008. Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence. J. Cell Biol. 180:101–112.
Sesaki, H., S.M. Southard, A.E. Hobbs, and R.E. Jensen. 2003. Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion. Biochem. Biophys. Res. Commun. 308:276–283.[CrossRef][Medline]
Sesaki, H., C.D. Dunn, M. Iijima, K.A. Shepard, M.P. Yaffe, C.E. Machamer, and R.E. Jensen. 2006. Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p. J. Cell Biol. 173:651–658.
Simbeni, R., F. Paltauf, and G. Daum. 1990. Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae. J. Biol. Chem. 265:281–285.
Steglich, G., W. Neupert, and T. Langer. 1999. Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria. Mol. Cell. Biol. 19:3435–3442.
Tatsuta, T., K. Model, and T. Langer. 2005. Formation of membrane-bound ring complexes by prohibitins in mitochondria. Mol. Biol. Cell. 16:248–259.
Tong, A.H., M. Evangelista, A.B. Parsons, H. Xu, G.D. Bader, N. Page, M. Robinson, S. Raghibizadeh, C.W. Hogue, H. Bussey, et al. 2001. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 294:2364–2368.
Voelker, D.R. 2004. Lipid synthesis and transport in mitochondrial biogenesis. In Topics in Current Genetics, vol. 8. Springer, Berlin/Heidelberg. 267–291.
Voelker, D.R. 2005. Bridging gaps in phospholipid transport. Trends Biochem. Sci. 30:396–404.[CrossRef][Medline]
Wang, X., X. Zuo, B. Kucejova, and X.J. Chen. 2008. Reduced cytosolic protein synthesis suppresses mitochondrial degeneration. Nat. Cell Biol. 10:1090–1097.[CrossRef][Medline]
Zhong, Q., V.M. Gohil, L. Ma, and M.L. Greenberg. 2004. Absence of cardiolipin results in temperature sensitivity, respiratory defects, and mitochondrial DNA instability independent of pet56. J. Biol. Chem. 279:32294–32300.
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100; data represent mean values ± SD of three independent experiments. (B, left) Wild type, 
