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Vol. 19, Issue 6, 2457-2464, June 2008
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*Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD 21702; and Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Submitted February 29, 2008;
Accepted March 7, 2008
Monitoring Editor: Janet Shaw
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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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; Sesaki and Jensen, 1999; Fritz et al., 2003; Okamoto and Shaw, 2005; Hoppins et al., 2007). Disruption of fission in the budding yeast Saccharomyces cerevisiae drives the equilibrium toward fusion resulting in net-like mitochondrial structures that appear to collapse to one side of the cell (fused). Conversely, abrogation of fusion shifts the equilibrium toward fission, which is manifest as dot-like fragmented mitochondria (fizzed; see examples in Figure 1A). Such alterations are linked to apoptosis in mammals and compromise cell life span in fungus (Okamoto and Shaw, 2005; Chan, 2006; Heath-Engel and Shore, 2006; Martinou and Youle, 2006; Scheckhuber et al., 2007). Maintaining the capacity of mitochondria to fuse normally is essential to inheritance of mitochondrial DNA and in turn for respiration (Hermann et al., 1998).
Mitochondrial outer membrane fusion is controlled by mitofusins, a family of GTPases integral to the mitochondrial outer membrane. The yeast mitofusin, Fzo1p, is unstable during both vegetative and nonvegetative growth. Regulating levels of expression of Fzo1p is of critical importance as either deletion or overexpression of Fzo1p alters the mitochondrial fusion process resulting in fizzed or abnormal aggregated mitochondria, respectively (Hermann et al., 1998; Rapaport et al., 1998; Fritz et al., 2003; Escobar-Henriques et al., 2006).
The ubiquitin-proteasome system (UPS) constitutes the major mechanism by which cells acutely alter levels of cytosolic, nuclear, and endoplasmic reticulum (ER) proteins in a highly regulated manner. This occurs generally, but not exclusively, by conjugation with chains of ubiquitin linked through lysine 48 (K48) of ubiquitin, which targets modified proteins to the 26S proteasome for degradation (Glickman and Ciechanover, 2002). Ubiquitin ligases (E3s) mediate the transfer of ubiquitin from ubiquitin-conjugating enzymes (E2s) to specific substrates and are the primary determinants of specificity in ubiquitylation (Fang and Weissman, 2004). During nonvegetative growth, corresponding to mating conditions, a role for the 26S proteasome in degradation of Fzo1p has been reported. However, evidence for ubiquitylation has surprisingly been lacking, and no ubiquitin ligase has been implicated in this process (Neutzner and Youle, 2005; Escobar-Henriques et al., 2006).
During vegetative growth, the level of Fzo1p is regulated by Mdm30p. Mdm30p is a member of the F-box family of proteins. The F-box is a 50-amino acid protein interaction motif encoded in 
15 genes in S. cerevisiae and 70 genes in mammals. F-box proteins are generally thought to serve as substrate recognition elements of ubiquitin ligases of the Skp1-Cullin-F-box (SCF) family (Cardozo and Pagano, 2004; Willems et al., 2004; Petroski and Deshaies, 2005). In this regard, Mdm30p has been shown to promote ubiquitylation and proteasomal degradation of the Gal4p transcription factor (Muratani et al., 2005). Nonetheless, to date not all F-box proteins have been positively linked to the SCF complex or even to a specific function in the UPS (Galan et al., 2001; Frescas et al., 2007).
Mdm30p associates with mitochondria (Fritz et al., 2003), appears to physically interact with Fzo1p (Escobar-Henriques et al., 2006), and has been shown to target Fzo1p for degradation (Fritz et al., 2003; Escobar-Henriques et al., 2006). The importance of Mdm30p in mitochondrial function is underscored by the finding that deletion of MDM30 abrogates mitochondrial fusion and leads to aggregated mitochondria (Dürr et al., 2006; see examples in Figure 1A) and consequently to defective mitochondrial DNA inheritance and a failure to respire (Fritz et al., 2003). However, the mechanism by which Mdm30p promotes Fzo1p degradation has not been elucidated.
In this study, we investigated the mechanism by which Fzo1p is targeted for degradation during vegetative growth. We establish that Fzo1p is ubiquitylated and targeted for proteasomal degradation. This ubiquitylation is mediated by an SCF ubiquitin ligase that includes Mdm30p as the substrate recognition factor (SCFMdm30p). Thus, we have identified a mechanism whereby a critical protein integral to the mitochondrial outer membrane is targeted for destruction by cytosolic components of the UPS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the indicated strains (see Supplemental Data, Table S1), FZO1 was chromosomally tagged with three copies of an HA epitope sequence, as previously described (Longtine et al., 1998).
Plasmids
The plasmids used in this study are listed in Supplemental Data, Table S1. Ubiquitin wild type, K48R, or K63R, expressed under the control of the CUP1 promoter were overproduced by growing cells for 1 h in the presence of 0.1 mM copper sulfate. The MDM30-hemagglutinin (HA) construct was generated as follows. The 500 base pairs upstream of the ATG and downstream of the STOP codon of MDM30 were subcloned in the pRS316 vector, yielding the pRS316 MDM30prom/ter vector. MDM30 lacking its STOP codon or both its STOP codon and sequence encoding its F-box (amino acids 1-58) were cloned into pRS316 MDM30prom/ter followed by insertion of the HA tag downstream of the MDM30 coding sequence, resulting in MDM30-HA and 
fbox-HA, respectively. MDM30-HA was then subcloned in the p426TEF vector and previously described mutations of amino acids in its F-box motif (fbox-HA; Escobar-Henriques et al., 2006) that are critical for its association with Skp1p (Galan and Peter, 1999) were generated by site-directed mutagenesis.
Antibodies
Antiserum raised against Fzo1p was generously provided by J. Nunnari (University of California, Davis, CA). Monoclonal antibodies utilized were against HA and Myc epitopes (12CA5 and 9E10; Santa Cruz Biotechnology, Santa Cruz, CA) phosphoglucokinase (PGK; Monoclonal 22C5; Molecular Probes, Eugene, OR), Tom20p (kindly provided by A. Azem, Tel Aviv University), Hexokinase1 (Hxk1p; kindly provided by A. Azem), or Cdc53p (yC-17; Santa Cruz Biotechnology). Antiserum recognizing Cue1p was provided by Z. Kostova (National Cancer Institute).
Yeast Extracts and Cycloheximide Chase
Cells grown in YPD or SD were collected during the exponential growth phase. Total protein extracts were prepared by the NaOH-trichloroacetic acid (TCA) lysis method (Avaro et al., 2002). To monitor constitutive turnover of Fzo1, cycloheximide (CHX) was added to yeast cultures growing at 37°C to a final concentration of 100 µg/ml. Thermosensitive strains were incubated for 2 h at 37°C before adding CHX. Total protein extracts were prepared at the indicated time points after addition of CHX.
Pulse-Chase Metabolic Labeling
Cells grown in YPD were collected during the exponential growth phase, incubated for 50 min at 23°C in 1 ml labeling media (SD-Met) lacking methionine, and pulsed with 25 µCi of 35S methionine (Perkin-Elmer Cetus, Norwalk, CT) per OD of cells for 15 min at 30°C followed by 15 min at 37°C. After addition of 1 ml chase media (SD-Met supplemented with 6 mg/ml methionine and 2 mg/ml BSA), cells were incubated at 37°C. After addition of chase media 2 ODs of cells were collected immediately (time = 0) and at 30 and 60 min and treated as described previously (Moreau et al., 1997). Results were quantified by Storm Phosphorimager and ImageQuant software (GE Healthcare, Waukesha, WI). Error bars were determined by calculating the SD from three independent experiments.
Immunoprecipitation
For coimmunoprecipitation assays, cells were lysed at 4°C with glass beads in immunoprecipitation (IP) buffer (50 mM HEPES, pH 7.5, 50 mM sodium chloride, 0.6% Triton X-100, 10% glycerol, 20 mM iodoacetamide, and protease inhibitor; Complete mini, Roche, Indianapolis, IN). Insoluble material was removed by centrifugation for 30 min at 13,000 x g. An aliquot of the supernatant was precipitated with 50% TCA (pre-IP lysate). The remaining supernatant was incubated with Anti-HA Affinity Matrix (Roche) for 2 h at 4°C. Beads washed with IP buffer were heated in sample buffer before resolution by SDS-PAGE and analysis by immunoblotting. For immunoprecipitation of Fzo1-HA or Mdm30-HA, yeast extracts were prepared using the NaOH-TCA lysis method (Avaro et al., 2002). Extracts were then boiled for 10 min at 70°C in SDS loading buffer and insoluble material removed by centrifugation for 5 min at 13,000 xg. Resulting supernatants were diluted 10-fold in IP buffer, and immunoprecipitations were performed as described above.
Subcellular Fractionation
Cell fractionation was performed as described (Meisinger et al., 2000). In brief, cell cultures were grown to midlog phase (OD6001-2), and spheroplasts were prepared by treating the cells with Zymolyase-20T (MP Biomedicals, Solon, OH). After gentle homogenization of spheroplasts and centrifugation at 1500 x g, the supernatant (total fraction) was further subject to centrifugation at 12,000 x g for 10 min, yielding a supernatant (S) and a mitochondrial enriched pellet fraction (P). Subcellular fractions were assayed for cytosolic and mitochondrial proteins; Tom20 and Hexokinase1 were used as mitochondrial and cytosolic markers, respectively.
Glycerol Growth Analysis
For glycerol growth assays, cultures grown overnight in SD medium were pelleted, resuspended at OD600 = 1, and diluted 1:10 five times in water. Three microliters of the dilutions were spotted on plates and grown for 2 d (SD) or 4 d (SG) at 30°C or 37°C.
Microscopy
For visualization of mitochondria, yeast strains were transformed with plasmid pYX232-mtGFP, encoding mitochondria-targeted GFP (mtGFP; Westermann and Neupert, 2000). Cultures in logarithmic growth were fixed with 3.7% formaldehyde (Sigma, St. Louis, MO) for 10 min, washed in KPi buffer (0.02 M KH2PO4, 0.08 M K2HPO4, 1 M sorbitol, pH 7.5) and mounted on Superfrost microscope slides (Esco Products, Oak Ridge, NJ) in phosphate-buffered saline. Cells were then analyzed by epifluorescence microscopy on an Axiovert 200M microscope (Carl Zeiss MicroImaging, Thornwood, NY) using a 100x oil-immersion objective. Images were recorded with a Hamamatsu ORCA-ER camera (Hamamatsu Photonics, Hamamatsu City, Japan). For each field of cells, between 30 and 40 pictures were taken in the Z-coordinate, and cells were deconvoluted using Improvision Openlab 4.0.2 software (Improvision, Lexington, MA). The morphology of mitochondria was assessed by counting more than 300 cells per strain. Quantification was confirmed by independent counting by a second individual blinded to the identity of the strains. Results are displayed in Supplemental Data Table S2 and Figure 1B.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cells expressing either wild-type Mdm30p or a form mutated in its F-box (Figure 1, B and C, and Supplemental Data, Table S1). This mutation has previously been shown to result in increased steady-state levels of Fzo1p (Escobar-Henriques et al., 2006). About 70% of wild-type cells displayed mitochondria characterized as being tubular. The remaining 30% were partitioned between those scored as having fused, fizzed, and aggregated mitochondria (see examples in Figure 1A). In agreement with previous reports (Fritz et al., 2003; Dürr et al., 2006; Escobar-Henriques et al., 2006), among mdm30 cells almost 70% displayed aggregated mitochondria (Figure 1C, vector transformation). Reintroducing MDM30 into mdm30 cells (MDM30-HA), under a constitutive TEF promoter, restored mitochondrial morphology to a distribution similar to wild-type cells and completely reversed the marked increase in aggregated mitochondria seen in mdm30 cells (Figure 1C). However, cells expressing the F-box mutant (fbox-HA) retained a mitochondrial distribution similar to that observed in the mdm30 mutant, pointing to an essential role for the F-box in maintaining a normal distribution of mitochondrial morphology.
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cells to respire properly and a decreased capacity to grow on media containing only a nonfermentable carbon source (Fritz et al., 2003; Dürr et al., 2006). Strains from Figure 1C were therefore tested for growth on selective media containing either dextrose (fermentable) or glycerol (nonfermentable) as the sole carbon source (Figure 1D). As shown previously (Fritz et al., 2003), mdm30 cells are defective for growth on glycerol media. Consistent with the morphological findings, this growth defect was rescued by expression of Mdm30-HA but not the F-box mutant Mdm30-HA (fbox-HA), once again pointing to an essential role for the F-box motif in mitochondrial function as well as morphology. Similar findings for both mitochondrial morphology and growth on glycerol were obtained when wild-type and truncated Mdm30p lacking its F-box (f-box-HA) were expressed from the endogenous MDM30 promoter (Supplemental Data, Figure S1).
Mitochondrial aggregation in mdm30 cells has been shown to correlate with accumulation of Fzo1p (Fritz et al., 2003). More recently, Mdm30p was shown to be essential for Fzo1p degradation during vegetative growth (Escobar-Henriques et al., 2006). In agreement with this latter study, we observed that endogenous Fzo1p, which is naturally turned over in cells, was completely stabilized upon genomic deletion of MDM30 (Figure 1E, left panels). Given our findings that the F-box of Mdm30p is required for normal mitochondrial morphology and respiration (Figure 1, C and D), we asked whether the F-box is also required for Fzo1p degradation. mdm30 strains expressing Mdm30-HA or its F-box mutant (fbox-HA) were used to monitor Fzo1p turnover (Figure 1E, right panels). Although Fzo1p was degraded upon expression of Mdm30-HA, it remained stable upon expression of the F-box mutant (fbox-HA), consistent with previous findings (Escobar-Henriques et al., 2006). This differential stability was also observed with wild-type and F-box–deleted Mdm30p expressed from its endogenous promoter (Supplemental Data, Figure S1, C and D). Together, results from Figure 1 indicate that the F-box of Mdm30p is essential for efficient mitochondrial fusion and respiration as well as for Fzo1p degradation.
Critical Components of the SCF Complex Participate in Fzo1p Degradation
Most F-box proteins are thought to act as substrate recognition subunits of SCF ubiquitin ligase complexes (Cardozo and Pagano, 2004; Willems et al., 2004; Petroski and Deshaies, 2005), thereby promoting substrate ubiquitylation followed by proteasomal degradation. In S. cerevisiae Skp1p serves as an adaptor between F-box proteins and the cullin, Cdc53p. The Skp1p-Cdc53p core together with a small RING finger protein serves as a molecular scaffold that also recruits a specific ubiquitin-conjugating enzyme (E2), Cdc34p.
Our finding that the F-box of Mdm30p is essential for Fzo1p degradation raises the possibility that Mdm30p functions as part of an SCF E3 ubiquitin ligase (SCFMdm30p), potentially providing a mechanism by which Mdm30p targets Fzo1p for degradation. To directly assess whether Mdm30p associates with Skp1p-Cdc53p in cells, HA-tagged Mdm30p was immunoprecipitated from whole cell extracts prepared from mdm30 cells expressing wild-type Mdm30p (Mdm30-HA) or Mdm30p mutated in its F-box (fbox-HA). Immunoprecipitates were resolved on SDS-PAGE and immunoblotted with Cdc53p antibody (Figure 2A, middle panel). Cdc53p coimmunoprecipitated with wild-type Mdm30-HA but not the F-box mutant. This result establishes that Mdm30p associates with core components of the SCF and that this interaction is dependent on an intact Mdm30p F-box.
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30 min in wild-type cells, it was markedly stabilized in thermosensitive mutants of either CDC53 or CDC34 (cdc53ts and cdc34ts) with half-lives of greater than 60 min. These results establish that, in addition to Mdm30p, critical components of the SCF complex participate in Fzo1p degradation.
Fzo1p Is Modified with Ubiquitin at the Mitochondria
The observation that the ubiquitin ligase SCFMdm30p is required for Fzo1p degradation strongly suggests that Fzo1p is a substrate for ubiquitylation. Ubiquitylated intermediates are frequently difficult to detect as they are rapidly degraded by the proteasome. Using a polyclonal antibody directed against Fzo1p, we observed multiple immunoreactive species above the major Fzo1p band on long exposure of immunoblots (Figure 3A, right panel). These higher molecular weight bands were reproducibly and specifically observed in extracts prepared from wild-type yeast but not from fzo1 cells. The same pattern was observed whether monitoring endogenous Fzo1p or chromosomally tagged FZO1-HA using either anti-Fzo1p or anti-HA (Figure 3B). A slight retardation in the migration pattern of Fzo1p and the higher molecular weight bands was observed in the FZO1-HA strain when detected by Fzo1p antibody, consistent with increased mass conferred by the three copies of an HA tag. This indicates that these higher molecular weight species represent modified forms of Fzo1p.
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As Fzo1p is an integral mitochondrial outer membrane protein, we wanted to determine the cellular location of ubiquitylated Fzo1p. Whole cell extract was fractionated and cytosolic (S) and mitochondria-enriched fractions (P) were tested for Fzo1p content by immunoblotting with Fzo1p antibody (Figure 3E). Both unmodified and ubiquitylated forms of Fzo1p were found almost exclusively in the mitochondria-enriched fraction. The mitochondrial localization of ubiquitylated Fzo1p was further confirmed by sucrose gradient analysis (Supplemental Data, Figure S2). These data indicate that ubiquitylated Fzo1p is localized to mitochondria and does not represent mis-targeted or mis-localized Fzo1p.
SCFMdm30p Mediates K48-linked Ubiquitylation of Fzo1p
Having established that the higher molecular weight species of Fzo1p correspond to Fzo1p-ubiquitin conjugates, we asked whether ubiquitylation of Fzo1p is dependent on Mdm30p and other SCF components. As is apparent, the ubiquitylated forms of Fzo1p that were detected in wild-type cells were undetectable in the mdm30 mutant (Figure 4A, cf. left two lanes). Moreover, although MDM30-HA restored Fzo1p ubiquitylation in mdm30 cells, the F-box mutant (fbox-HA) did not. Similarly, Fzo1p ubiquitylation was decreased in both conditional SCF mutants, cdc34ts and cdc53ts, when grown at the restrictive temperature compared with isogenic wild-type controls (Figure 4B). We conclude that Mdm30p, Cdc34p, and Cdc53p are essential for the Fzo1p ubiquitylation that is observed in wild-type cells.
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). Other cellular functions are known to involve monoubiquitylation or polyubiquitylation through other lysines of ubiquitin, especially K63 (Mukhopadhyay and Riezman, 2007). To gain insight into the linkages being generated on Fzo1p, wild-type and fzo1 cells were transformed with a high copy vector for overexpression of ubiquitin (wild type) or ubiquitin in which either K48 or K63 was mutated to arginine (K48R or K63R). Overexpression of wild-type ubiquitin is known to enhance the steady-state level of substrate ubiquitylation. Incorporation of overexpressed K48R or K63R ubiquitins into chains precludes elongation of polyubiquitin chains linked through these residues (Galan and Haguenauer-Tsapis, 1997). Strikingly, we observed that overexpression of K48R ubiquitin resulted in a downward shift in ubiquitylated forms of Fzo1p, reflecting either multiple mono-ubiquitylation events or short chains "capped" with unextendable K48R ubiquitin (Figure 4C). In contrast, overexpression of wild-type or K63R ubiquitin resulted in an upward shift in ubiquitylated species of Fzo1p, consistent with increased availability of ubiquitin competent for K48 chain elongation. Taken together, these data not only confirm that Fzo1p is ubiquitylated, but also provide strong evidence that the higher molecular weight forms include modification with K48-linked polyubiquitin chains.
Fzo1p Is Degraded by the 26S Proteasome during Vegetative Growth
The results presented thus far establish that Fzo1p is ubiquitylated at the mitochondria in a manner that is dependent on an intact SCFMdm30p ubiquitin ligase, that this ubiquitylation appears to be largely K48-linked in nature, and that this modification strongly correlates with degradation of Fzo1p, because deletion of MDM30 or mutation of its F-box abolishes both ubiquitylation and degradation of Fzo1p. Collectively these observations raise the possibility that Fzo1p turnover during vegetative growth is a consequence of proteasomal degradation.
To test the effect of altered proteasome function on Fzo1p degradation, Fzo1p turnover was assayed by CHX chase in two different proteasome mutant strains (cim3-1 and pre1-1 pre2-2), which we compared with their wild-type isogenic controls. The conditional proteasome mutations either slowed (pre1-1 pre2-2) or completely inhibited (cim3-1) degradation of Fzo1p (Figure 5A). These results strongly suggest that Fzo1p is a target for the ubiquitin-proteasome system.
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Finally, steady-state levels of unmodified and ubiquitylated Fzo1p were assayed in yeast strains bearing thermosensitive mutations in 26S proteasome subunits from the 19S lid (mpr1-1), 19S base (rpt2RF, cim3-1) or 20S core (pre1-1 pre2-2). In each of these four examples, steady-state levels of unmodified as well as ubiquitylated Fzo1p increased in proteasome mutants relative to the four different wild-type isogenic control strains at 37°C but not at 23°C (Figure 5C, cf. right and left panels). These results confirm the importance of the proteasome in Fzo1p degradation and provide further evidence that ubiquitylated forms of this protein are targeted for degradation. Together, the results presented in Figure 5 establish a clear role for proteasomes in regulating the constitutive turnover of Fzo1 during vegetative growth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of reports have provided indirect links between the UPS and mitochondria (Fisk and Yaffe, 1999; Sutovsky et al., 1999; Hitchcock et al., 2003; Peng et al., 2003; Thompson et al., 2003; Rinaldi et al., 2004; Altmann and Westermann, 2005; Dürr et al., 2006). The best evidence so far for involvement of the UPS in directly regulating mitochondrial outer membrane proteins is in mammals where a specific E3, MARCHV/ MITOL, is implicated in ubiquitylating two components of the mitochondrial fission apparatus, DRP1 and FIS1, which are the human orthologues of yeast Dnm1p and Fis1p, respectively. However, there is a lack of consensus as to whether this ubiquitylation serves to target these factors for proteasomal degradation or facilitates other nonproteolytic functions (Nakamura et al., 2006; Yonashiro et al., 2006; Karbowski et al., 2007).
The literature regarding the mitochondrial mitofusins is even more complicated. During nonvegetative (mating type) growth, degradation of the single yeast mitofusin, Fzo1p, has been suggested to be dependent on the proteasome. However, there is no direct evidence for ubiquitylation or involvement of a specific E3, and a role for Mdm30p has been excluded (Neutzner and Youle, 2005).
The groups of Westermann and Langer have demonstrated, and we confirm herein, that during vegetative growth, degradation and maintenance of normal levels of Fzo1p is dependent on the F-box protein Mdm30p (Fritz et al., 2003), with a specific requirement for an intact F-box (Escobar-Henriques et al., 2006). However, Escobar-Henriques et al. concluded that this degradation of Fzo1p is independent of ubiquitin, the SCF and proteasomes (Escobar-Henriques et al., 2006). This led the authors to conclude that Fzo1p is degraded during vegetative growth by a novel UPS-independent proteolytic pathway that was still dependent on Mdm30p having an intact F-box. The findings presented in the current study lead to a very different conclusion. We provide direct evidence of ubiquitylation of endogenously expressed Fzo1p that is highly suggestive of K48-linked ubiquitin chains. This ubiquitylation is dependent on Mdm30p capable of assembling with other SCF components through its F-box motif. Moreover, both CHX and 35S pulse-chase metabolic labeling experiments implicate the UPS and particularly SCFMdm30p in Fzo1p degradation. Our internally consistent positive findings unequivocally establish an important role for the UPS in determining the fate of Fzo1p. The discrepancy between the conclusions reached in Escobar-Henriques et al. and our findings are difficult to reconcile. However, we certainly cannot discount the possibility that in addition to the UPS other means of degrading Fzo1p could exist including through mitochondrial and other cytosolic proteases as well as by autophagy. Regardless, the clear involvement of the UPS in Fzo1p degradation, established herein, should lay the groundwork for further investigation of the signals that target Fzo1p for degradation and the significance of this degradation in mitochondrial function.
The findings presented in this study are in accord with a paradigm based on cells lacking Mdm30p (Fritz et al., 2003): increased levels of Fzo1p due to failure to regulate the level of this protein results in mitochondrial aggregation and a failure to respire. Despite the requirement for the Mdm30p F-box in Fzo1p degradation found in Escobar-Henriques et al., the same group found that Mdm30 lacking its F-box overexpressed from the CUP promoter surprisingly restored normal mitochondrial morphology in mdm30 cells (Dürr et al., 2006). In contrast, we find that, in these cells, expression of F-box mutants of Mdm30p from either the TEF or MDM30 promoter results in a failure to reverse the abnormal aggregated mitochondrial morphology seen in mdm30 cells, a finding confirmed by the failure to restore growth on a nonfermentable carbon source (glycerol). As suggested (Dürr et al., 2006), the restoration of mitochondrial morphology obtained with the CUP promoter in Dürr et al. could be a consequence of overexpression of truncated Mdm30 that binds to and inactivates excess Fzo1p. If this is the case, it is unlikely of physiological significance.
The determination that an integral membrane protein of the mitochondrial outer membrane is ubiquitylated while still mitochondria-associated and unambiguously degraded by the proteasome leads us to posit a general UPS-dependent process of mitochondrial-associated degradation (MAD). Analogous to ERAD, ubiquitin ligases intrinsic to the mitochondrial outer membrane, such as MARCHV/ MITOL (Nakamura et al., 2006; Yonashiro et al., 2006; Karbowski et al., 2007) and the newly described MULAN (Li et al., 2008), as well as E3s that are recruited to the mitochondrial outer membrane, such as the SCF, will be involved in this process. As with ERAD there is likely to be a high degree of complexity, and we predict that a number of mitochondrial and cytosolic proteins will be implicated in playing roles either in protecting proteins or facilitating their targeting to the UPS and in retro-translocation from mitochondrial membranes through as yet to be established mechanisms. With definitive proof for involvement of the UPS at the mitochondria now established, a number of exciting questions arise including how individual substrates are recognized; the extent to which the UPS might be involved in the fate of proteins in the intramembranous space, the inner mitochondrial membrane, and the mitochondrial matrix; and the degree to which MAD targets misfolded proteins as well as highly regulated normal proteins involved in critical mitochondrial functions such as fusion and fission.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Michael H. Glickman (glickman{at}technion.ac.il) or Allan M. Weissman (amw{at}nih.gov)
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