TY - JOUR
AU - Esaki, Masatoshi
AB - Abstract Mitochondria continuously undergo coordinated fusion and fission during vegetative growth to keep their homogeneity and to remove damaged components. A cytosolic AAA ATPase, Cdc48, is implicated in the mitochondrial fusion event and turnover of a fusion-responsible GTPase in the mitochondrial outer membrane, Fzo1, suggesting a possible linkage of mitochondrial fusion and Fzo1 turnover. Here, we identified two Cdc48 cofactor proteins, Ubp3 and Ubx2, involving mitochondria regulation. In the absence of UBP3, mitochondrial fragmentation and aggregation were observed. The turnover of Fzo1 was not affected in Δubp3, but instead a deubiquitylase Ubp12 that removes fusion-required polyubiquitin chains from Fzo1 was stabilized. Thus, excess amount of Ubp12 may lead to mitochondrial fragmentation by removal of fusion-competent ubiquitylated Fzo1. In contrast, deletion of UBX2 perturbed disassembly of Fzo1 oligomers and their degradation without alteration of mitochondrial morphology. The UBX2 deletion led to destabilization of Ubp2 that negatively regulates Fzo1 turnover by removing degradation-signalling polyubiquitin chains, suggesting that Ubx2 would directly facilitate Fzo1 degradation. These results indicated that two different Cdc48-cofactor complexes independently regulate mitochondrial fusion and Fzo1 turnover. Cdc48/p97, cofactor, membrane fusion, mitochondria, ubiquitin A protein family called as ATPases associated with diverse cellular activities (AAA) is found in all kingdoms of life. Most of AAA proteins facilitate unfolding of substrate proteins, thereby disassembling protein and protein-DNA complexes and degrading the substrates. Cdc48, also called as p97, VCP and TER94, is an essential AAA protein located in cytosol and nucleus and is highly conserved in all eukaryotes. Cdc48 is one of the most abundant intracellular proteins, which ensure its multiple cellular functions including the ubiquitin-proteasome dependent protein degradation system (1, 2), homotypic fusion of endoplasmic reticulum (ER) and Golgi membranes (3–6), nuclear envelope reassembly (7), ER-associated protein degradation (8–11), autophagy (12–14), protection against protein aggregation (15), mitochondria-associated protein degradation (16–18) and mitochondrial morphology regulation (19–21). These diverse cellular functions of Cdc48 are achieved by two types of cofactors, i.e. substrate-recruiting cofactors that interact with substrate proteins such as ubiquitin and bring them to Cdc48, and substrate-processing cofactors that modulate substrate proteins before and after processing by Cdc48 and deliver to downstream factors such as the proteasome (22–25). Thus, the cofactors qualify Cdc48 for specific subcellular localizations, specific cellular functions and specific substrates (26). In this study, we report Cdc48 cofactors regulating mitochondrial morphology and mitochondrial protein turnover. Mitochondria, double-membrane organelles, are organized into tubular networks with controlled balance between fusion and fission of mitochondrial membranes (27, 28). It is conceivable that the mitochondrial plasticity contributes to relief of damages caused by reactive oxygen species and to elimination of severely damaged mitochondria by autophagy. Abnormal regulation of mitochondrial morphology lies behind several neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Dynamin-related large GTPases on the mitochondrial outer and inner membranes play essential roles on the membrane fusion and fission. Yeast Fzo1, which is a homologue of mitofusins in mammals, on the mitochondrial outer membrane is the GTPase responsible for the outer membrane fusion (29, 30). Upon mitochondrial outer membrane fusion, Fzo1 functions as a tethering complex by forming a trans-oligomer with other Fzo1 molecules on apposed mitochondrion (21, 31, 32). Ubiquitylation of Fzo1 as well as GTP hydrolysis is required for the membrane fusion and for quality control of the GTPase. Two distinct types of ubiquitylation on Fzo1, which are removed by two different deubiquitylases, Ubp2 and Ubp12, are reported to have opposing effects on mitochondrial fusion (31). The Ubp12-specific ubiquitylation of Fzo1 is prerequisite for mitochondrial fusion, while the Ubp2-specific ubiquitylation of Fzo1 facilitates Fzo1 degradation, thereby inhibiting the fusion. Cdc48 was shown to interact with ubiquitylated Fzo1, but much less efficiently with unmodified Fzo1 (20). This is analogous to other Cdc48-regulated cellular reactions, in which ubiquitylated proteins are the major substrates of Cdc48. However, the fate of the ubiquitylated Fzo1 after recognition by Cdc48 is controversial since some groups reported that Cdc48-deficiency resulted in stabilization of Fzo1 (19, 33, 34) whereas another group showed destabilization of Fzo1 in a Cdc48 mutant (20). Nevertheless, it has reached consensus that loss-of-function of Cdc48 led to deficiency of mitochondrial fusion (19, 20, 35), suggesting that Cdc48 facilitates mitochondrial outer membrane fusion through regulation of Fzo1. Ubp12 was also shown to receive ubiquitylation and to be a substrate of Cdc48 for degradation (20). Likewise, Ubp2 received ubiquitylation, which led to degradation (36). Thus, Cdc48- and ubiquitin-dependent cascades for Fzo1, Ubp2 and Ubp12 are intricately intertwined to regulate mitochondrial fusion. As mentioned above, Cdc48 functions with its cofactors. One of the Cdc48 cofactors, Vms1, was reported to be required for Fzo1 degradation under a specific condition (33), but not during vegetative growth (19). It was also shown that Vms1 accumulated on mitochondria in response to abnormal co-translational translocation (37). Another Cdc48 cofactor, Doa1, was reported to be required for degradation of several ubiquitylated proteins on the mitochondrial outer membrane, including Mcl1 (an anti-apoptotic protein of the Bcl2 family), Tom70 (a component of the protein translocator) and Fzo1 (34). However, lack of Doa1 did not phenocopy Cdc48 deficiency on mitochondrial morphology; the deletion of the chromosomal DOA1 gene slightly enhanced mitochondrial tubular network (34), whereas Cdc48 deficiency led to accumulation of fragmented and aggregated mitochondria (19, 20). Therefore, other Cdc48 cofactors likely function in regulation of mitochondrial morphology. Cdc48 cofactors can be defined by possession of Cdc48-interacting motifs such as ubiquitin regulatory X (UBX), VCP-interacting motif (VIM) and peptide: N-glycanase and UBA or UBX-containing proteins (PUB) (38, 39). The budding yeast Saccharomyces cerevisiae genome contains around 20 Cdc48 cofactor genes. In this study, we employed comprehensive analysis to identify Cdc48 cofactors affecting mitochondrial morphology and Fzo1 regulation. Materials and Methods Yeast strains and growth media All yeast strains used in this study were derivatives of a haploid strain W303a. Strains expressing mitochondria-targeting green fluorescent protein (mito-GFP) were generated by transformation with pAK42 (40). Cdc48 cofactor-deleted strains were constructed by replacement of the whole coding region of the chromosomal genes with the Candida glabrata HIS3 and TRP1 genes (National BioResource Project, Japan). All strains were grown at 30°C in a minimal medium containing 2% glucose with appropriate supplements (41). Plasmids The coding regions of UBX2 and UBP3 including 500 bp 5′-untranslated region and 300 bp 3′-untranslated region were amplified by PCR, and cloned into pRS313 (42). Fluorescence microscopy Cells expressing mito-GFP were visualized by fluorescence and differential interference contrast (DIC) microscopy without chemical fixation. Stacks of optical sections were captured by laser confocal microscopy (Leica TCS SP2 and SP8), and 2 D images were generated with maximum projection. Sucrose density gradient centrifugation Cells were grown in the minimal medium containing 2% glucose at 30°C until an OD600 reached around 0.5. Mitochondria were isolated as described previously (43). Isolated mitochondria (∼500 µg) were incubated with 1% (w/v) digitonin in 500 µl of a lysis buffer composed of 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, protease inhibitor cocktail (Nacalai Tesque, Inc.) for 20 min at 4°C. Insoluble materials were removed by centrifugation at 15,000 × g for 10 min at 4°C, and the supernatants were loaded on the top of a 12 ml 5–20% (w/v) linear sucrose gradient in the lysis buffer with 0.1% digitonin, which had been prepared using Gradient Master (SK BIO International, Co. Ltd.). The gradients were centrifuged at 174,000 × g for 14 h at 4°C. The gradients were fractionated into 32 aliquots of 400 μl by collecting from the top of the gradients. The proteins were precipitated with trichloroacetic acid and analysed by SDS-PAGE and immunoblotting. Blots were quantified using Typhoon Trio+ and Imagequant TL (GE Healthcare). Results Ubp3 is involved in mitochondrial morphology regulation—among ∼20 Cdc48 cofactors in S.cerevisiae, only Ufd1 and Npl4 are indispensable for vegetative growth (44, 45). Therefore, we constructed a yeast strain collection in which non-essential Cdc48 cofactors were deleted by replacing the whole open reading frame in the chromosome with a marker gene (46). When visualized using mito-GFP, a few mitochondrial tubules in wild type (WT) cells were observed under vegetative growth in glucose-containing media (Fig. 1A and B). Since Cdc48 deficiency led to accumulation of fragmented mitochondria due to fusion defect (19, 35), we distinguished cells showing punctate and aggregated fluorescence. Most of the deletion mutants including Δdoa1 and Δvms1 predominantly showed normal tubular mitochondrial network like the WT strain (Fig. 1A and B) (19). In contrast, the deletion of UBP3 resulted in fragmented and aggregated mitochondria (Fig. 1A and B), which resembles to those observed in Cdc48-deficient strains (19). Introduction of a single-copy plasmid expressing Ubp3 under the control of its own promoter rescued the mitochondrial defective morphology in Δubp3 (Fig. 1C and D). To assess the possibility that deletion of Cdc48 cofactors affect overall mitochondrial functions, we tested mitochondrial oxidative phosphorylation ability. Growth on media with a non-fermentable carbon source such as lactate requires mitochondrial respiration to produce adenosine triphosphate (ATP) by oxidative phosphorylation. Lack of Bcs1, a AAA protein in the mitochondrial inner membrane, failed to assemble the respiratory complex III, resulting in growth defect in a non-fermentable carbon source (47). By contrast, most of Cdc48 cofactor-deleted strains showed no obvious growth differences in fermentable glucose and non-fermentable lactate media except for Δubp3 and Δubx2 showing a limited respiratory growth defect (Fig. 2). Fig. 1 View largeDownload slide Mitochondrial morphology of wild type (WT) and Cdc48 cofactor-deleted strains. (A) Cdc48 cofactor-deleted cells transformed with a plasmid expressing mitochondria-targeting GFP were grown to early exponential growth phase in a glucose medium at 30°C. Live cells were directly examined by fluorescent microscopy. The scale bar indicates 5 μm. (B) Proportions of cells exhibiting tubular (grey bars) and punctate/aggregated (black bars) fluorescence were determined from 100–300 cells. (C) Mitochondrial morphology in Δubp3 cells with a Ubp3-expressing plasmid was analysed using mitochondria-targeting GFP. (D) Numbers of cells exhibiting tubular (grey bars) and punctate/aggregated (black bars) fluorescence were counted. Fig. 1 View largeDownload slide Mitochondrial morphology of wild type (WT) and Cdc48 cofactor-deleted strains. (A) Cdc48 cofactor-deleted cells transformed with a plasmid expressing mitochondria-targeting GFP were grown to early exponential growth phase in a glucose medium at 30°C. Live cells were directly examined by fluorescent microscopy. The scale bar indicates 5 μm. (B) Proportions of cells exhibiting tubular (grey bars) and punctate/aggregated (black bars) fluorescence were determined from 100–300 cells. (C) Mitochondrial morphology in Δubp3 cells with a Ubp3-expressing plasmid was analysed using mitochondria-targeting GFP. (D) Numbers of cells exhibiting tubular (grey bars) and punctate/aggregated (black bars) fluorescence were counted. Fig. 2 View largeDownload slide Growth of Cdc48 cofactor-deleted mutants in non-fermentable carbon source media. Cells grown in a glucose medium were diluted in 10-fold increments and 3 μl of cells from each dilution (starting with 0.5 OD600 units) were spotted on minimal medium plates supplemented with 2% glucose or lactate, and incubated for 2–4 days at 30 and 37°C. Fig. 2 View largeDownload slide Growth of Cdc48 cofactor-deleted mutants in non-fermentable carbon source media. Cells grown in a glucose medium were diluted in 10-fold increments and 3 μl of cells from each dilution (starting with 0.5 OD600 units) were spotted on minimal medium plates supplemented with 2% glucose or lactate, and incubated for 2–4 days at 30 and 37°C. Fzo1 turnover is perturbed in Δubx2—in the Cdc48-deficient cells, protein stability of the fusion-responsible GTPase Fzo1 was affected. We therefore tested whether Cdc48 cofactors contributes to Fzo1 stability. The steady state level of Fzo1 in Δubp3 was markedly upregulated whereas Δdoa1 led to slight downregulation of Fzo1 (Fig. 3A and B). The abnormal high Fzo1 level in Δubp3 could not be rescued by the plasmid expressing Ubp3 (Fig. 3F and G) probably due to low expression of Ubp3 that is enough to suppress defect in mitochondrial morphology, but not in Fzo1 protein abundancy. Next, we assessed the turnover rates of Fzo1 by addition of cycloheximide to inhibit new protein synthesis. The deletion of neither UBP3 nor DOA1 significantly affected the turnover rate of Fzo1 (Fig. 3A and C). In contrast, Fzo1 turnover was markedly retarded in a deletion mutant of another Cdc48 cofactor Ubx2. Turnover rates of a mitochondrial outer membrane protein Tom40 and Cdc48 itself were mostly indistinguishable among the WT strain and the Cdc48-cofactor-deleted strains tested, although a slightly faster degradation of Cdc48 was observed in Δubp3 (Fig. 3A, D and E). Expression of endogenous level of Ubx2 rescued the defective turnover rate of Fzo1 in Δubx2 (Fig. 3G and H). Because Δubx2 showed no mitochondrial morphology defect (Fig. 1), mitochondrial fragmentation observed in Δubp3 and reduced degradation rate of Fzo1 observed in Δubx2 seem to be independent. Fig. 3 View largeDownload slide Stability and turnover of proteins. (A) Cells expressing Fzo1-HA (19) were grown to early exponential growth phase in a glucose-containing medium. After addition of 100 µg/ml cycloheximide (CHX) to inhibit new protein synthesis, the cells were further incubated at 30°C. Whole cell lysates were prepared at indicated time as described previously (19), and analysed by SDS-PAGE followed by immunoblotting using anti-HA, Cdc48 and Tom40 antibodies. Immunoblotting was visualized with Typhoon Trio+ and quantified with Imagequant TL (GE Healthcare). (B) Steady state levels of Fzo1-HA were determined by band intensities at time 0 in each strains. Averages and standard deviations of three independent experiments were shown. The amount in the wild type cells was set to 100%. *P≤0.05; **P≤0.01. (C–E) Band intensities of Fzo1-HA (C), Cdc48 (D) and Tom40 (E) at indicated time points were quantified, and the averages with standard deviations calculated from three independent experiments were plotted. The protein amounts in the wild type cells were set to 100%. (F) The Δubx2 and Δubp3 cells were transformed with single-copy plasmids expressing Ubx2 and Ubp3, respectively. Protein stabilities of Fzo1-HA, Cdc48 and Tom40 after inhibition of new protein synthesis were analysed as described in (A). (G, H) Band intensities of Fzo1-HA at each time point were plotted as in (B) and (C). Fig. 3 View largeDownload slide Stability and turnover of proteins. (A) Cells expressing Fzo1-HA (19) were grown to early exponential growth phase in a glucose-containing medium. After addition of 100 µg/ml cycloheximide (CHX) to inhibit new protein synthesis, the cells were further incubated at 30°C. Whole cell lysates were prepared at indicated time as described previously (19), and analysed by SDS-PAGE followed by immunoblotting using anti-HA, Cdc48 and Tom40 antibodies. Immunoblotting was visualized with Typhoon Trio+ and quantified with Imagequant TL (GE Healthcare). (B) Steady state levels of Fzo1-HA were determined by band intensities at time 0 in each strains. Averages and standard deviations of three independent experiments were shown. The amount in the wild type cells was set to 100%. *P≤0.05; **P≤0.01. (C–E) Band intensities of Fzo1-HA (C), Cdc48 (D) and Tom40 (E) at indicated time points were quantified, and the averages with standard deviations calculated from three independent experiments were plotted. The protein amounts in the wild type cells were set to 100%. (F) The Δubx2 and Δubp3 cells were transformed with single-copy plasmids expressing Ubx2 and Ubp3, respectively. Protein stabilities of Fzo1-HA, Cdc48 and Tom40 after inhibition of new protein synthesis were analysed as described in (A). (G, H) Band intensities of Fzo1-HA at each time point were plotted as in (B) and (C). Fzo1 assembly is affected in Δubx2– Fzo1 was shown to form homo-oligomers and hetero-oligomers with other outer membrane proteins such as Ugo1 (48, 49). Fzo1 oligomer formation and disassembly are prerequisite for the membrane fusion and Fzo1 turnover. We next tested whether the Cdc48 cofactors affect Fzo1 oligomerization. Mitochondria were isolated from vegetatively growing cells by the conventional differential centrifugation method, and solubilized with a non-ionic detergent digitonin. The solubilized protein complexes were analysed by sucrose density gradient centrifugation. Fzo1 in WT mitochondria predominantly formed a single peak (Fig. 4A). On the other hand, Fzo1 in Δubx2 was recovered in a more-dense fraction as well as the similar fraction of WT (Fig. 4A). We verified the reliability of the assay by checking that the Tom40 assembly was not affected by deletion of the Cdc48 cofactor (Fig. 4B). Deletion of UBP3 did not markedly affect the Fzo1 oligomerization (Fig. 4C). These results suggest that Ubx2, but not Ubp3, is responsible for Fzo1 disassembly. Fig. 4 View largeDownload slide Complex formation of Fzo1-HA. Mitochondria were isolated from exponentially growing wild type, Δubx2 (A, B) and Δubp3 (C) cells expressing Fzo1-HA. Isolated mitochondria were solubilized with 1% digitonin and analysed by sucrose density gradient centrifugation. The gradients were then fractionated into 32 samples and analysed by SDS-PAGE followed by immunoblotting using anti-HA and anti-Tom40 antibodies. Amounts of Fzo1-HA (A, C) and Tom40 (B) recovered in the fraction 1–18 were shown because no proteins were essentially detected in the fraction 19–32. Total amounts of the proteins recovered in all fractions were set to 100%. Ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (156 kDa), catalase (232 kDa) and ferritin (470 kDa) were used as molecular weight standards. Fig. 4 View largeDownload slide Complex formation of Fzo1-HA. Mitochondria were isolated from exponentially growing wild type, Δubx2 (A, B) and Δubp3 (C) cells expressing Fzo1-HA. Isolated mitochondria were solubilized with 1% digitonin and analysed by sucrose density gradient centrifugation. The gradients were then fractionated into 32 samples and analysed by SDS-PAGE followed by immunoblotting using anti-HA and anti-Tom40 antibodies. Amounts of Fzo1-HA (A, C) and Tom40 (B) recovered in the fraction 1–18 were shown because no proteins were essentially detected in the fraction 19–32. Total amounts of the proteins recovered in all fractions were set to 100%. Ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (156 kDa), catalase (232 kDa) and ferritin (470 kDa) were used as molecular weight standards. Stability of deubiquitylases Ubp2 and Ubp12 is also affected by the Cdc48 cofactors—Ubp2 and Ubp12 are involved in mitochondrial fusion and Fzo1 turnover by removal of different ubiquitin chains on Fzo1. In addition, these two deubiquitylases also receive ubiquitylation, and the ubiquitylated Ubp12, and possibly Ubp2 as well, are subject to Cdc48-dependent degradation (20). We then tested whether degradation of Ubp2 and Ubp12 is affected by deletion of the Cdc48 cofactors. C-terminally HA-tagged Ubp2 and Ubp12 were expressed in expected endogenous levels. Ubp12-HA was substantially accumulated in the absence of UBP3 as compared to that in the WT cells although turnover rates of Ubp12-HA in Δubp3 and Δubx2 were quite similar to the WT level (Fig. 5A, B and C). By contrast, we observed apparent destabilization of Ubp2-HA in Δubx2 (Fig. 5D, E and F), indicating that Ubx2 leads to stabilization of Ubp2 to some extent. Because Cdc48-cofactor complexes mostly lead to degradation of their substrate proteins, Ubx2 may not directly regulate Ubp2 but instead Ubx2 may facilitate degradation of upstream factors required for Ubp2 degradation such as the ubiquitin E3 ligase Mdm30 (36). Taken these results together, two Cdc48 cofactors, Ubp3 and Ubx2, regulate different steps regarding mitochondria-related protein stability and mitochondrial morphology. Fig. 5 View largeDownload slide Protein stability of Ubp2-HA and Ubp12-HA. Steady state levels and turnover of Ubp12-HA (A–C) and Ubp2-HA (D–F) were analysed as described in Fig. 1. Averages and standard deviations of three independent experiments were shown. *P≤0.05. Fig. 5 View largeDownload slide Protein stability of Ubp2-HA and Ubp12-HA. Steady state levels and turnover of Ubp12-HA (A–C) and Ubp2-HA (D–F) were analysed as described in Fig. 1. Averages and standard deviations of three independent experiments were shown. *P≤0.05. Discussion Cofactor proteins define specific cellular functions of Cdc48. Here we showed that Ubp3 and Ubx2 are involved in mitochondria-relating functions of Cdc48. Ubp3 forms a deubiquitylase complex with Bre5 (50). Ubp3 was shown to regulate protein quality control, vesicular transport, the Ras signalling pathway and osmotic shock-responsive gene expression (50–53). Ubp3 was also shown to inhibit mitochondria-specific autophagy called mitophagy, but promote other autophagy including ribosomal autophagy (54, 55). Ubp3 predominantly localizes in the cytosol under normal growth conditions whereas induction of mitophagy strongly accumulates Ubp3 on mitochondria (54). In this study, we showed that deletion of UBP3 led to abnormal mitochondrial morphology and accumulation of Fzo1 and Ubp12. Similar accumulation of Fzo1 was observed in Δotu1 independently of fragmented mitochondria. Fragmented mitochondrial morphology, but not accumulation of Fzo1, in Δubp3 was recovered by expression of Ubp3 from a single-copy plasmid. These results suggest that the Fzo1 accumulation may not be the reason for abnormal mitochondrial morphology observed in Δubp3 although overexpression of Fzo1 was shown to lead to fragmentation of mitochondria (56). Instead, it is reasonable to assume that polyubiquitin chains of Ubp12, which is subject to Cdc48-mediated degradation (20), may be removed by the Ubp3 deubiquitylase complex. The resulting accumulation of Ubp12 could remove the fusion-competent polyubiquitin chains of Fzo1, which accounts for abnormal mitochondrial morphology in Δubp3. It is also possible that Ubp3, like Ubp12, directly interact with Fzo1 and remove the fusion-competent polyubiquitin chains. Ubx2 is an integral membrane protein and predominantly localizes to the ER for clearance of misfolded proteins in the ER (57, 58). Ubx2 was also shown to be required for clearance of model substrate proteins accumulated in the cytosol and the nucleus (1, 57). Δubx2 showed sensitivity to cadmium that induces oxidative stress response of mitochondria (1, 59), implying a Ubx2 function related to mitochondria. A proteomic analysis showed that some mitochondrial proteins such as an outer membrane protein Tom6 were stabilized in Δubx2 (60). Consistently, we also found in this study that Fzo1 turnover was retarded in Δubx2. By contrast, deletion of UBX2 led to Ubp2 destabilization. Ubp2 was shown to remove the degradation-signalling polyubiquitin chains of Fzo1 and deletion of UBP2 led to extremely low stability of Fzo1 (31), suggesting that reduction of the Ubp2 abundancy in Δubx2 should facilitate Fzo1 degradation. Nevertheless, deletion of UBX2 led to stabilization of Fzo1, suggesting that Ubx2 may function at a later step than the Ubp2-specific step in the Fzo1 degradation pathway. We also showed that Δubx2 accumulated an apparent larger complex of Fzo1, which would be homo-oligomers or hetero-oligomers with another outer membrane protein Ugo1 (49). Deletion of UGO1 led to disassembly of Fzo1 oligomers and degradation of Fzo1 (48, 56, 61), suggesting that the oligomers perturb degradation of Fzo1. As Cdc48 can function as a segregase, the Cdc48-Ubx2 complex may be involved in the disassembly process of Fzo1 oligomers prior to degradation. Because the Ubp2-specific ubiquitylation of Fzo1 could occur independently of oligomer formation of Fzo1 (31), disassembly/degradation and deubiquitylation seem not to be correlated, and it is interesting to reveal what molecular features and/or signals determine the fate of Fzo1. Fzo1 is thought to function as a tethering complex for fusion by trans-oligomer formation with other Fzo1 molecules on apposed mitochondrion. It is obvious that needless Fzo1 trans-oligomers should be eliminated following membrane fusion. The turnover rate of Fzo1 was not perturbed when mitochondrial fusion was defective in Δubp3, suggesting that degradation of the Fzo1 trans-oligomer does not greatly contribute to mitochondrial fusion. This is consistent with a previous observation that fusion-competent ubiquitylation of Fzo1 did not facilitate degradation of Fzo1 (31). Digitonin-solubilized Fzo1 oligomers accumulated in Δubp3 were mostly indistinguishable from those in WT mitochondria probably because population of the trans-oligomer is too low to be detected, although it is possible that digitonin solubilization could not preserve the Fzo1 trans-oligomer. We could not observe any obvious ubiquitylated forms of Fzo1, Ubp2 and Ubp12, which had been shown to be hardly detectable (20, 31). Restricted expression of deubiquitylases may improve such a detection limit. In this study, we showed two Cdc48 cofactors, Ubp3 and Ubx2, affect mitochondrial protein behaviours. Otu1 and Vms1 have also been implicated in mitochondria at least under certain conditions (33, 34, 37). Differential contributions and intermolecular relationships of these Cdc48 cofactors are interesting to be elucidated in the future. Acknowledgements We thanks Dr. Toshiya Endo for providing a plasmid expressing mitochondria-targeting GFP, staffs of Liaison Laboratory Research Promotion Center of the Institute of Molecular Embryology and Genetics, Kumamoto University, for their technical assistances and our lab members for valuable comments. Funding This work was supported by KAKENHI grants (#24770189 and #15K07007) the Program for Leading Graduate Schools HIGO (Health life science: Interdisciplinary and Glocal Oriented) from MEXT, Japan, and in part by KAKENHI grants (#24370056 and #16H04764) from JSPS, the CREST program JPMJCR13M1 from JST. Conflict of Interest None declared. References 1 Schuberth C. , Richly H. , Rumpf S. , Buchberger A. 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( 2009 ) A mutation associated with CMT2A neuropathy causes defects in Fzo1 GTP hydrolysis, ubiquitylation, and protein turnover . Mol. Biol. Cell. 20 , 5026 – 5035 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AAA ATPases associated with diverse cellular activities ER endoplasmic reticulum mito-GFP mitochondria-targeting green fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Two Cdc48 cofactors Ubp3 and Ubx2 regulate mitochondrial morphology and protein turnover
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvy057
DA - 2018-06-19
UR - https://www.deepdyve.com/lp/oxford-university-press/two-cdc48-cofactors-ubp3-and-ubx2-regulate-mitochondrial-morphology-Sdwox7070b
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -