Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion
Mitochondria are dynamic organelles constantly undergoing fusion and fission events , modulated by a variety of post-translational modifiers including ubiquitin ( Escobar-Henriques and Langer , 2014; Komander and Rape , 2012 ) . Due to their pathological relevance , e . g . for Parkinson’s disease , these processes are subject to intense investigation . For example , Parkin-dependent ubiquitylation of mitochondrial outer membrane ( OM ) proteins modulates the elimination of the damaged organelles by mitophagy , or via mitochondrial-derived vesicles ( MDV ) that fuse with the late endosome ( Pickrell and Youle , 2015; Sugiura et al . , 2014 ) . Most fusion processes , including the Parkin-MDV pathway , rely on SNAREs ( McLelland et al . , 2016 ) . In contrast , fusion of the endoplasmic reticulum ( ER ) and of mitochondria depend on large dynamin-related GTPases ( Escobar-Henriques and Anton , 2013; Hu and Rapoport , 2016 ) . In mitochondria , they are named mitofusins ( Mfn1/Mfn2 in mammals , Fzo1 in yeast ) . Deficiencies in Mfn2 cause the type 2 subset of the Charcot-Marie-Tooth disease ( CMT ) , the most common degenerative disorder of the peripheral nervous system ( Züchner et al . , 2004 ) . The ubiquitin-specific chaperone Cdc48/p97 is required to maintain mitochondrial morphology ( Esaki and Ogura , 2012 ) . However , the underlying molecular mechanism of how Cdc48 regulates mitochondrial dynamics is not understood . Cdc48 is an essential AAA-ATPase and one of the most abundant proteins in the cell , which recognizes many ubiquitylated substrates and is involved in a myriad of biological processes ( Franz et al . , 2014; Meyer and Weihl , 2014 ) . Cdc48 segregates ubiquitylated substrates from protein complexes , or from membranes , thus allowing their proteolysis by the proteasome ( Franz et al . , 2014 ) . For example , Cdc48 is important for ER-associated protein degradation ( ERAD ) , modulates the turnover of mitochondrial OM proteins ( OMMAD ) , participates in apoptosis responses ( Laun et al . , 2001 ) and mediates clearance of damaged lysosomes by autophagy ( Avci and Lemberg , 2015; Heo et al . , 2010; Papadopoulos et al . , 2017; Tanaka et al . , 2010; Wu et al . , 2016; Xu et al . , 2011; Zattas and Hochstrasser , 2015 ) . On the other hand , Cdc48 also binds E3 ubiquitin ligases and deubiquitylases ( DUBs ) thereby regulating substrate ubiquitylation ( Meyer and Weihl , 2014 ) . DUBs are proteases that catalyze the reversion of the ubiquitylation reaction ( Love et al . , 2007 ) , critically contributing to ubiquitin homeostasis ( Amerik and Hochstrasser , 2004; Kimura and Tanaka , 2010; Park and Ryu , 2014; Swatek and Komander , 2016 ) . DUBs activate ubiquitin by releasing it from ubiquitin precursor polypeptides but are also determinants for the modification status of ubiquitylated substrates , allowing to dampen ubiquitin-mediated events ( Clague et al . , 2013 ) . Importantly , DUBs are associated with a number of human diseases and represent promising drug targets , whose regulation and mechanism of action need to be explored ( Heideker and Wertz , 2015; Sahtoe and Sixma , 2015 ) . Two deubiquitylases , Ubp2 and Ubp12 , were found to have opposite effects on mitochondrial morphology ( Anton et al . , 2013 ) . Ubiquitin chains on Fzo1 that are recognized and cleaved by Ubp12 activate mitochondrial fusion . In contrast , other ubiquitin chains on Fzo1 that instead are recognized and cleaved by Ubp2 target Fzo1 for proteasomal degradation and inhibit mitochondrial fusion . Therefore , although it is clear that ubiquitin is a double-faced regulator of mitochondrial fusion ( Escobar-Henriques and Langer , 2014 ) , how Ubp2 and Ubp12 exert opposite effects on Fzo1 and mitochondrial fusion remained poorly studied . Here , we identify a role of Cdc48 in mitochondrial fusion , as part of a novel enzymatic cascade consisting of Cdc48 , Ubp12 and Ubp2 . Cdc48 negatively regulates Ubp12 , which negatively regulates Ubp2 , explaining why these two DUBs exert opposite effects on their targets and on ubiquitin homeostasis .
Although it is clear that Cdc48 affects mitochondrial dynamics ( Esaki and Ogura , 2012 ) , the underlying mechanisms are unclear . The role of Cdc48 for mitochondrial morphology was investigated in the hypomorphic mutant cdc48-2 , expressing GFP targeted to mitochondria . In this allele , Cdc48 is mutated for A547T , in its ATPase domain D2 , whereas in the most commonly used cdc48-3 strain , Cdc48 is instead mutated in R387K , in the D1 ATPase ( C . Hickey and M . Hochstrasser , p . communication ) . Both cdc48-3 and cdc48-2 mutations impair typical Cdc48-dependent processes for transmembrane proteins , like ERAD ( Bays et al . , 2001; Hitchcock et al . , 2001; Latterich et al . , 1995 ) . We observed that cdc48-2 cells presented fragmented mitochondria ( Figure 1A ) , consistent with the mitochondrial phenotypes observed upon impairment of the ATPase activity of Cdc48 ( Esaki and Ogura , 2012 ) . This suggested problems in mitochondrial fusion and prompted us to evaluate the role of Cdc48 on Fzo1 , present at the outer membrane of mitochondria . Mitochondrial fusion is abolished in the absence of Fzo1 ubiquitylation ( Anton et al . , 2013 ) . Consistent with mitochondrial fragmentation , we observed a decrease of Fzo1 ubiquitylation in cdc48-2 mutant cells , when compared to wild-type ( wt ) cells ( Figure 1B , black arrows ) . We have previously shown that pro-fusion ubiquitylation of Fzo1 increases its stability ( Anton et al . , 2013 ) . Accordingly , the steady state levels of Fzo1 and its ubiquitylated forms were decreased in cdc48-2 cells ( compare Figure 1C and B ) , to a similar and not significantly different extent ( data not shown ) . Consistent with the cdc48-2 allele , the levels of Fzo1 were slightly decreased in the cdc48-3 mutant or in cells deleted for the Cdc48 co-factors Npl4 , Ufd1 and Ufd3/Doa1 ( Figure 1—figure supplement 1A–C ) . It was previously shown that Ubc6 , an endoplasmic reticulum ( ER ) membrane protein , is degraded by the proteasome via ERAD , a process dependent on Cdc48 ( Lenk et al . , 2002 ) . Therefore , we also analyzed the steady state levels of Ubc6 in the same CDC48 mutant strains . As expected , and in contrast to Fzo1 , the steady state levels of Ubc6 were increased upon impairment of Cdc48 activity ( Figure 1C and Figure 1—figure supplement 1A–C ) . This suggested that Cdc48 regulates Fzo1 by a mechanism different from OMMAD or ERAD . Since both Fzo1 and Ubc6 were mostly affected in the cdc48-2 mutant , we decided to use this strain for further analysis . However , it is unclear why cdc48-2 affects Ubc6 and Fzo1 stronger than cdc48-3 . We investigated why cdc48-2 mutant cells have lower levels of Fzo1 , by testing with cycloheximide ( CHX ) chase experiments if Cdc48 regulates Fzo1 stability . Moreover , to simultaneously test the role of the proteasome , we deleted the efflux pumps Snq2 and Pdr5 . We observed that Fzo1 degradation was inhibited by the presence of the proteasome inhibitor MG132 , indicating that the decreased levels of Fzo1 observed in cdc48-2 cells were due to proteasome-dependent turnover of Fzo1 ( Figure 1D ) . In contrast , proteasome inhibition did not affect Fzo1 turnover in wt cells consistent with previous observations ( Anton et al . , 2013; Escobar-Henriques et al . , 2006 ) . Importantly , all these phenotypes could be rescued by expression of the wt Cdc48 protein but not by expression of the Cdc48A547T variant , mimicking the specific mutation in cdc48-2 ( Figure 1—figure supplement 2A–C ) . In conclusion , Cdc48 is required to maintain the Fzo1 protein , thus promoting mitochondrial fusion events . We further investigated how Cdc48 affected Fzo1 . Given that stress conditions disrupt mitochondrial tubulation ( Knorre et al . , 2013 ) , it was important to show that Cdc48 directly regulates Fzo1 and mitochondrial morphology . First , co-immunoprecipitation experiments revealed that Cdc48 physically interacted with Fzo1 ( Figure 2A ) . We previously showed that the formation of ubiquitin chains on Fzo1 ( Figure 2A , black arrows ) , which are linked to lysine 398 , requires previous ubiquitylation of its lysine 464 ( Anton et al . , 2013 ) . Therefore , Fzo1 ubiquitylation is lost in the mutant Fzo1K464R ( Figure 2A ) . We observed that the interaction between Cdc48 and the non-ubiquitylated variant Fzo1K464R was impaired ( Figure 2A ) , in agreement with ubiquitin being recognized by Cdc48 . To assess the specificity of the cdc48-2 effect on Fzo1 protein levels , we tested if this depended on Fzo1 ubiquitylation . Thus , the non-ubiquitylated variant Fzo1K464R was used . We observed that the steady state levels of Fzo1K464R were largely insensitive to the cdc48-2 mutation ( Figure 2—figure supplement 1 ) . This points to a direct regulatory role of Cdc48 on Fzo1 , only after its ubiquitylation . These pro-fusion ubiquitin forms on Fzo1 are recognized by Ubp12 . In addition , we previously identified other ubiquitin forms on Fzo1 , that inhibit fusion . They are removed by Ubp2 and can be detected only in the presence of the catalytically inactive variant Ubp2C745S ( Anton et al . , 2013 ) ( Figure 2B , Input , red arrows ) . Therefore , we investigated binding of Cdc48 to Fzo1 under these conditions , where both pro-fusion and anti-fusion forms are present . We noticed that despite the clear increase in ubiquitylation of Fzo1 upon Ubp2C745S expression ( 2 . 44 times ) , Cdc48 binding to Fzo1 was not increased ( Figure 2B ) . Therefore , the additional presence of ubiquitin chains inhibiting fusion does not increase Cdc48 binding . Consistently , for the Fzo1K464R variant , which in the presence of Ubp2C745S is ubiquitylated to a similar level as the wt protein ( 0 . 96 times , despite the absence of pro-fusion ubiquitylation ) , no binding to Cdc48 above background can be detected . Thus , similar to Ubp12 , Cdc48 recognizes specifically the pro-fusion ubiquitylated forms of Fzo1 . Given the specific interaction of both Cdc48 ( Figure 2B ) and Ubp12 ( Anton et al . , 2013 ) with ubiquitin chains on Fzo1 promoting fusion , we tested if Cdc48 regulated Ubp12 . To analyze if Ubp12 is an unstable protein , wt and cdc48-2 cells were transformed with an episomal plasmid expressing Ubp12 under the ADH1 promoter ( Anton et al . , 2013 ) . CHX chase experiments revealed that Ubp12 is degraded in a Cdc48- and proteasome-dependent manner ( Figure 3—figure supplement 1A and B ) . Similarly , chromosomally tagged Ubp12 is an unstable protein and its turnover depends on Cdc48 ( Figure 3A ) . To analyze if Ubp12 is ubiquitylated , the DUB was immunoprecipitated and analyzed by immunoblotting for Ubp12-Flag or for ubiquitin ( Figure 3B ) . We observed slower migrating forms of Ubp12 with the Flag-specific antibody , which were also detected by a ubiquitin-specific antibody . These studies demonstrated that Ubp12 is modified by ubiquitin . We next tested whether Cdc48 could be co-immunoprecipitated with Ubp12 , from solubilized crude mitochondrial extracts . We observed that Ubp12 physically interacted with Cdc48 ( Figure 3C ) , suggesting that Cdc48 directly supports degradation of ubiquitylated Ubp12 . Our results show that Cdc48 and Ubp12 have opposing roles on Fzo1 ubiquitylation levels ( Figure 1B and [Anton et al . , 2013] ) . Consistently , Ubp12 and Cdc48 also present opposing phenotypes regarding mitochondrial tubulation ( Figure 1A and [Anton et al . , 2013] ) . Given that Cdc48 controls Ubp12 levels , we speculated that Cdc48 regulates mitochondrial morphology and Fzo1 via Ubp12 . We monitored mitochondrial morphology in cdc48-2 cells in presence or absence of UBP12 , expressing mitochondrial-targeted GFP . Strikingly , deletion of UBP12 in cdc48-2 cells rescued mitochondrial tubulation , resembling Δubp12 cells ( Figure 4A ) . Importantly , the mitochondrial hypertubulation of Δubp12 cells depended on Fzo1 ( Figure 4—figure supplement 1A–C ) . Even in Δfzo1 Δdnm1 cells , resembling wt cells in mitochondrial shape , further deletion of UBP12 did not induce hypertubulation , confirming that Ubp12 regulates mitochondrial morphology via Fzo1 ( Figure 4—figure supplement 1D ) . Mitochondrial fusion is also required to maintain the cellular growth on respiratory media , i . e . media containing the non-fermentable carbon sources glycerol or lactate ( Hermann et al . , 1998 ) . Therefore , to further support the physiological importance of Cdc48 and Ubp12 , we analyzed the respiratory capacity of cdc48-2 in presence or absence of UBP12 . In agreement with restored tubulation of mitochondria , we observed that the growth defect of cdc48-2 cells at 37°C on lactate media could be improved upon deletion of UBP12 ( Figure 4B ) . Given that Δfzo1 cells irreversibly loose mitochondrial DNA , we investigated if this is also the case for cdc48-2 cells . Consistent with the respiratory reversibility of cdc48-2 cells upon further deletion of UBP12 , we observed that cdc48-2 cells did not lose mitochondrial DNA ( Figure 4—figure supplement 2A and B ) . Importantly , the respiratory defect of cdc48-2 cells could be complemented by expression of Cdc48 but not of Cdc48A547T ( Figure 4—figure supplement 2C ) . Finally , cdc48-2Δubp12 cells also showed improved ubiquitylation of Fzo1 ( Figure 4C ) . Together , these results show that Cdc48 maintains Fzo1 ubiquitylation and activates mitochondrial fusion by downregulating Ubp12 . However , two pieces of evidence suggest that Cdc48 might have other functions in this pathway , apart from regulating Ubp12 . First , we observed that the physical interaction between Fzo1 and Cdc48 is not mediated by Ubp12 ( Figure 4—figure supplement 2D ) , suggesting that Cdc48 directly recognizes ubiquitylated Fzo1 . Second , deletion of UBP12 in cdc48-2 cells did not restore the steady state levels of Fzo1 ( Figure 4—figure supplement 2E ) . Notably , this is consistent with our previous observation that mitochondrial fusion depends on ubiquitylated rather than on the steady state levels of Fzo1 ( Anton et al . , 2013 ) . We noticed that increased levels of Fzo1 , present in ∆ubp12 cells , specifically depended on Ubp2 ( Figure 5A ) . Therefore , Ubp12 and Ubp2 , which affect the stability of Fzo1 in opposite manners , are also interdependent . Next , we analyzed if Ubp2 and Ubp12 also presented other opposing and interdependent phenotypes related to ubiquitin . First , we analyzed cellular growth of cells lacking UBP2 , UBP12 or both , in the presence of sub-lethal doses of CHX , a phenotype commonly tested to monitor imbalances in ubiquitin homeostasis ( Gerlinger et al . , 1997; Hanna et al . , 2003; Rumpf and Jentsch , 2006 ) . Second , we directly quantified the levels of free ubiquitin vs . substrate-conjugated ubiquitin in the same strains . We observed that indeed Ubp2 and Ubp12 had opposite phenotypes ( Figure 5—figure supplement 1 ) . In addition , the consistent interdependence of these two enzymes suggested a DUB hierarchy , which prompted us to test a possible regulation of the Ubp2 protein by Ubp12 . We tested if Ubp2 is an unstable protein and whether Ubp12 is involved in its degradation , after inhibition of protein synthesis with CHX . The levels of genomically tagged Ubp2 decreased over time and Ubp2-turnover was regulated by Ubp12 ( Figure 5B ) and by the proteasome ( Figure 5—figure supplement 2A ) . Moreover , co-immunoprecipitation experiments revealed that Ubp2 interacted with Ubp12 , suggesting a direct regulation between both DUBs ( Figure 5—figure supplement 2B ) . We therefore investigated if Ubp2 could be ubiquitylated , in a Ubp12-dependent manner . After immunoprecipitation of Ubp2-Flag , and consistent with recent observations ( Cavellini et al . , 2017 ) , we observed the presence of slowly migrating forms of Ubp2 during electrophoresis , in wt cells ( Figure 5—figure supplement 2C ) but mostly in Δubp12 cells ( Figure 5C , left panel ) . Importantly , we show that these forms could also be detected using a ubiquitin-specific antibody , demonstrating that they represent ubiquitylated Ubp2 ( Figure 5C and Figure 5—figure supplement 2C , right panels ) . This indicates that Ubp12 mediates deubiquitylation of Ubp2 and suggests that Ubp2 acts downstream of Ubp12 , thus revealing a hierarchical cascade between DUBs , of relevance for the protein levels of Fzo1 and for ubiquitin homeostasis . In contrast to numerous proteins that are destabilized in absence of DUBs , deletion of UBP12 stabilizes Fzo1 ( Figure 6—figure supplement 1 ) and Ubp2 ( Figure 5B ) . Consistently , the two other known substrates of Ubp12 – Rad23 ( Gödderz et al . , 2017 ) and Gpa1 ( Wang et al . , 2005 ) are also not destabilized in Δubp12 cells . To characterize the deubiquitylation reaction of Ubp12 in more detail , we analyzed the ubiquitin linkages on Fzo1 and Ubp2 accumulating in Δubp12 cells . Overexpression of ubiquitin mutated in K48R strongly decreased Fzo1 and Ubp2 ubiquitylation , revealing that their ubiquitin chains are linked via K48 ( Figure 6A and C ) . However , the ubiquitin chains on Fzo1 that destabilize it and inhibit fusion , which are not bound by Ubp12 , are also K48-linked ( Figure 6B ) ( Anton et al . , 2013 ) . Thus , differences in ubiquitin chains cannot explain why Ubp12 stabilizes its substrates . To further analyze Ubp12 , its ubiquitin chain preference was tested using in vitro deubiquitylation assays ( Hospenthal et al . , 2015 ) . As a substrate , we used either K48-linked or K63-linked ubiquitin , present in the form of either di-ubiquitin ( Figure 6D ) or ubiquitin chains ( Figure 6E ) . However , in all cases , Ubp12 revealed no chain preference ( Figure 6D , E ) . This suggested that it is not Ubp12 but rather the chains themselves on the substrates that prevent their turnover . Thus , we determined the number of ubiquitin moieties present on Fzo1 , upon co-expression of tagged and non-tagged ubiquitin molecules . We observed that co-expression of ubiquitin and Myc-ubiquitin decomposed the first ubiquitylated form of Fzo1 , i . e running closest to non-modified Fzo1 , into two bands ( Figure 6F ) . This corresponds to the presence of either ubiquitin or Myc-ubiquitin attached to Fzo1 and confirms that this form corresponds to mono-ubiquitylated Fzo1 . Interestingly , however , for the two other ubiquitylated forms with lower electrophoretic mobility , we observed that only two additional bands could be observed above each of them . They correspond to either the presence of two Myc-ubiquitin molecules or one ubiquitin and one Myc-ubiquitin conjugated to Fzo1 . These results suggest that the K48 chains on Fzo1 consist of two ubiquitin moieties . In conclusion , Ubp12 recognizes ubiquitylated chains on Fzo1 composed of a very small number of ubiquitin moieties . We therefore propose that Ubp12 does not stabilize its substrates because their ubiquitin chains are too short to target proteasomal turnover . Both Ubp12 and Ubp2 deubiquitylate Fzo1 , but they clearly bind different forms of ubiquitylated Fzo1 ( Anton et al . , 2013 ) . Ubp12 binds ubiquitylated forms of Fzo1 that stabilize Fzo1 and promote mitochondrial fusion . In turn , Ubp2 recognizes other ubiquitylated forms of Fzo1 , that instead signal Fzo1 turnover thus preventing mitochondrial fusion . Given that Ubp12 acts upstream of Ubp2 , we speculated that the pro-fusion ubiquitylated forms of Fzo1 , Ubp12-specific , would also precede its Ubp2-specific anti-fusion forms . This predicts an impairment of anti-fusion forms in the absence of pro-fusion forms . Therefore , as previously , the mutant Fzo1K464R was chosen as a tool , because it loses the pro-fusion ubiquitylation ( Figure 7A , inset , black arrows , compare lanes 1 and 2 ) . Moreover , as in Figure 2B , the catalytically-inactive Ubp2C745S protein was expressed additionally . This allows visualization of the Ubp2-specific anti-fusion forms as well ( Figure 7A , inset , red arrows , lane 3 ) , resulting in a massive increase in overall ubiquitylation of Fzo1 ( compare lanes 1 and 3 ) . As predicted by our hypothesis , much of this increase was lost when K464 was mutated to R ( compare lanes 3 and 4 ) . This shows that Ubp2-dependent ubiquitylation largely requires previous K464-dependent ubiquitylation . Therefore , pro-fusion ubiquitylation , which stabilizes Fzo1 , primes Fzo1 for the formation of anti-fusion ubiquitylation . These anti-fusion forms , instead , signal Fzo1 for proteasomal degradation , so that in Δubp2 cells Fzo1 is less abundant ( Anton et al . , 2013 ) . Taking this into consideration , the steady state levels of Fzo1 were used as a read-out for the presence of anti-fusion ubiquitylation on Fzo1 . We noticed that whereas the steady state levels of Fzo1 decreased by 91% inΔubp2 cells , as expected , the steady state levels of Fzo1K464R only decreased by 47% ( Figure 7B ) . This shows that Fzo1K464R is much less sensitive to the deletion of UBP2 than wt Fzo1 , consistent with a lower abundance of the anti-fusion ubiquitylation . To confirm this result , the levels of Fzo1 were also tested upon further deletion of MDM30 inΔubp2 cells , which encodes the E3 ligase-component responsible for pro-fusion ubiquitylation on Fzo1 ( Cohen et al . , 2008; Escobar-Henriques et al . , 2006; Fritz et al . , 2003 ) . Indeed , we could observe a rescue of Fzo1 steady state levels inΔubp2 Δmdm30 cells , confirming that pro-fusion precedes anti-fusion ubiquitylation on Fzo1 ( Figure 7C ) . We conclude that Ubp2-specific ubiquitylation of Fzo1 largely depends on Ubp12-specific ubiquitylation of Fzo1 , indicating a regulatory cascade of Ubp12 and Ubp2 on Fzo1 . To challenge the Cdc48-DUBs regulatory cascade , we first tested if the role of Cdc48 on Fzo1 steady state levels depended on Ubp2 and Ubp12 . Indeed , and in contrast to wt cells , in ∆ubp2 ∆ubp12 cells the steady state levels of Fzo1 were insensitive to further mutating Cdc48 ( Figure 8A ) . Moreover , ∆ubp2 cells and ∆ubp2 ∆ubp12 were similarly insensitive to the presence of the cdc48-2 allele ( Figure 8B ) , consistent with the UBP2 UBP12 epistasis results ( Figure 5A and Figure 5—figure supplement 1A and B ) . Next , we tested if overexpression of Ubp2 could rescue cdc48-2 phenotypes . This was to be expected because deletion of UBP12 rescues CDC48 mutant phenotypes but also leads to increased levels of Ubp2 . Consistently , mitochondrial tubulation was significantly improved under these conditions ( Figure 8C ) . Moreover , Ubp2 overexpression improved the growth defect of cdc48-2 cells on lactate media at the non-permissive temperature of 37°C , supporting the physiological impact of the Ubp2 levels in cdc48-2 cells ( Figure 8D ) . Therefore , the respiratory capacity of the cdc48-2 cells could be improved not only by UBP12 deletion but also by overexpression of Ubp2 . Finally , a physical interaction between Ubp2 and Cdc48 could be observed ( Figure 8—figure supplement 1 ) . Together our results highlight a model in which Cdc48 , Ubp12 and Ubp2 orchestrate a multilayered cascade regulation , culminating on Fzo1 ubiquitylation and mitochondrial fusion .
Cdc48 promotes degradation of Ubp12 , controlling Fzo1 ubiquitylation . Ubp12 prevents mitochondrial fusion by two means . On the one hand , it removes the ubiquitylation on Fzo1 that is required for fusion . On the other hand , it promotes degradation of Ubp2 . This leaves the anti-fusion ubiquitylation of Fzo1 unopposed , resulting in Fzo1 degradation . Therefore , by supporting turnover of Ubp12 , Cdc48 dually preserves mitochondrial fusion events . In contrast , when only a non-functional variant of the protein is present , as is the case in cdc48-2 cells , Cdc48 cannot protect the pro-fusion ubiquitylation of Fzo1 . In this case , the cascade will synergistically converge in degradation of Fzo1 and thus inhibition of mitochondrial fusion will occur . The interdependence between these two pathways contributes to a coordinated cellular decision by Cdc48 to either fuse mitochondria or instead prevent it by degrading Fzo1 . Moreover , the Cdc48-Ubp12-Ubp2 cascade allows fine-tuning of substrate ubiquitylation and modulation of the biological processes thereof , as exemplified for Fzo1 and mitochondrial fusion ( Figure 9 ) . Cdc48/p97 extracts ubiquitylated substrates from membranes , thus allowing their recognition and degradation by the proteasome ( Franz et al . , 2014; Rape et al . , 2001 ) . This is exemplified with the ER protein Ubc6 , and was also shown for mitochondrial OM proteins ( Neutzner et al . , 2007 ) , including mitofusins under damaging conditions ( Tanaka et al . , 2010 ) . Therefore , Cdc48/p97 and ubiquitin regulate mitochondrial fusion in both yeast and mammals . Moreover , eukaryotes present a similar ubiquitin pattern of mitofusins , suggesting that the new function of Cdc48 presented here could be conserved in mammals under non-damaging conditions . Mitochondrial fusion is a complex multistep process dependent on sequential events involving GTP binding and hydrolysis by Fzo1 , Fzo1 oligomerization and finally ubiquitylation of Fzo1 ( Anton et al . , 2011; Brandt et al . , 2016; Cohen et al . , 2011; Ishihara et al . , 2004 ) . Although it is clear that ubiquitin critically determines mitochondrial fusion events , the underlying mechanisms are largely unknown ( Anton et al . , 2013 ) . The DUBs Ubp12 and Ubp2 cleave different ubiquitylated forms of Fzo1 that either promote or repress mitochondrial fusion , respectively ( Anton et al . , 2013 ) . Here , given that Ubp12 regulates Ubp2 , we show that these two ubiquitylation pathways are connected . Consistently , on Fzo1 , Ubp12-specific ubiquitylation also precedes Ubp2-specific ubiquitylation . In fact , unopposed anti-fusion ubiquitylation , as it is the case in Δubp2 cells , disrupts mitochondrial tubulation . This renders the role of Ubp2 in mitochondrial dynamics quite clear , namely protecting mitochondrial fusion . In contrast , the need for a dedicated DUB that removes the pro-fusion ubiquitylation forms , i . e . the need for Ubp12 , remained unclear . Now , the Ubp12-Ubp2 cascade allows to understand the purpose of Ubp12 , solving the paradox of why inhibition of the pro-fusion ubiquitylation on Fzo1 is required: in fact , too much pro-fusion ubiquitylation also means too much anti-fusion ubiquitylation , a problem counteracted by the deubiquitylation activity of Ubp12 on Fzo1 . We conclude that this cascade ensures a tight control of Fzo1 ubiquitylation at levels sufficient to allow mitochondrial fusion but preventing unnecessary ubiquitylation that instead targets Fzo1 for proteasomal turnover . The cascade between Ubp12 and Ubp2 also allows revising recent results linking Ubp2 and Mdm30 ( Cavellini et al . , 2017 ) . Mdm30 catalyzes the formation of the pro-fusion ubiquitin forms on Fzo1 ( Cohen et al . , 2008 ) . The pro-fusion forms are bound and cleaved by Ubp12 , depend on lysine 464 of Fzo1 , and are essential for mitochondrial fusion ( Anton et al . , 2013 ) . As to the anti-fusion ubiquitin forms on Fzo1 , two types could now be observed: low molecular weight , K464-independent , anti-fusion ubiquitylation ( as seen in Figure 7A , lane 4 ) , consistent with previous results ( Anton et al . , 2013 ) , but mostly high molecular weight anti-fusion ubiquitylation , instead K464-dependent ( as seen in Figure 7A , lane 3 ) . This shows that the anti-fusion ubiquitin forms on Fzo1 largely depend on its pro-fusion forms . Therefore , it is not surprising that anti-fusion , Ubp2-specific , ubiquitylation on Fzo1 also largely depends on Mdm30 . Nevertheless , future studies are required to clarify if Mdm30 itself catalyzes the formation of this high molecular weight fraction of the anti-fusion ubiquitylation on Fzo1 . Moreover , it is clear that Mdm30 is not the ligase responsible for the anti-fusion low molecular weight forms on Fzo1 ( Anton et al . , 2013 ) , which therefore remains to be identified . Our results unravel for the first time a regulatory cascade of two DUBs , Ubp12 and Ubp2 , with opposing functions in ubiquitin homeostasis . A 20–40% depletion in ubiquitin levels leads to cellular growth defects under various stress conditions in yeast , to lethality or infertility in mice , and to neurological diseases like ataxia , gracile axonal dystrophy or Parkinson’s disease ( Kimura and Tanaka , 2010; Park and Ryu , 2014 ) . The level of free ubiquitin is adjusted to the cellular needs , and is critically regulated by deubiquitylase activity ( Chernova et al . , 2003; Swaminathan et al . , 1999 ) . Here , we reveal distinct roles of two DUBs - Ubp2 and Ubp12 - for the maintenance of ubiquitin homeostasis . Δubp12 cells are hyperresistant to cycloheximide ( CHX ) , a chemical inhibitor of protein translation . Similar observations were previously reported in proteasome mutants , with impaired proteolysis ( Gerlinger et al . , 1997 ) . Consistently , just like proteasome mutants , also Δubp12 cells accumulate conjugated ubiquitin , without affecting the levels of free ubiquitin . In turn , Δubp2 cells showed a 40% depletion of free ubiquitin and hypersensitivity to CHX , consistent with similar observations in strains presenting decreased free ubiquitin levels ( Hanna et al . , 2003 ) . Nevertheless , along with reduced free ubiquitin , deletion of UBP2 also clearly led to increased levels of ubiquitin conjugates , as observed upon DmUsp5 depletion in the fruit fly ( Kovács et al . , 2015 ) . In fact , the importance of free ubiquitin pools versus ubiquitin conjugates for cellular growth is not well understood . Our analysis of Δubp2 cells sheds light on this question , demonstrating that depletion of free ubiquitin is epistatic over the accumulation of ubiquitylated conjugates for cellular growth . What could justify the opposite behavior of Ubp2 and Ubp12 in ubiquitin homeostasis and substrate turnover ? The removal of ubiquitin from a substrate is generally expected to increase its stability , as observed for Fzo1 in Δubp2 cells . Consistently , Ubp2 appears as a general quality control deubiquitylase recognizing both K48- and K63-linked ubiquitin chains that signal for turnover , both by the UPS and by the lysosome ( Anton et al . , 2013; Fang et al . , 2016; Ho et al . , 2017; Silva et al . , 2015 ) . In contrast , the turnover of both Fzo1 and Ubp2 is decreased in Δubp12 cells . Moreover , Ubp12 does not stabilize Rad23 ( Gödderz et al . , 2017 ) and Gpa1 ( Wang et al . , 2005 ) , i . e . its two other known substrates . Ubp12 exhibits a broad substrate specificity in vitro recognizing both K48- and K63-linked chains , consistent with previous observations ( Schaefer and Morgan , 2011 ) . Thus , it is not Ubp12 but the substrate that behaves unexpectedly . Notably , the ubiquitin signals that accumulate in Fzo1 , Ubp2 , Rad23 and Gpa1 are all composed of a limited number of discrete bands , instead of the high molecular weight smear , typical for polyubiquitylated substrates . For Fzo1 , we find that Ubp12 recognizes ubiquitylated forms that only contain two ubiquitin moieties that are linked via K48 . We propose that the presence of a short number of ubiquitin molecules on the ubiquitin chains recognized by Ubp12 could explain why they do not serve as a good signal for proteasomal degradation . The protein Met4 was also shown to be ubiquitylated with a a limited number of discrete bands ( Flick et al . , 2004; Kuras et al . , 2002 ) . In this case , intramolecular association with a ubiquitin binding domain in Met4 shields the ubiquitin chains , thus preventing their elongation and protecting Met4 against proteasomal degradation ( Flick et al . , 2006; Tyrrell et al . , 2010 ) . How deubiquitylation is controlled is poorly understood . Our findings suggest that this involves ubiquitylation of the DUBs themselves , because both Ubp2 and Ubp12 are regulated by ubiquitylation . This consequently renders DUBs interdependent , as exemplified with Ubp12 being the DUB of Ubp2 . Interestingly , several examples in the literature illustrate a big diversity of DUB regulation ( Michel et al . , 2017 ) . Therefore , additional mechanisms to proteolysis for the atypical function of Ubp2 ubiquitylation can be proposed . For example , Ubp2 ubiquitylation could induce a conformational change favouring catalytic activity , as observed for the DUB ATXN3 ( Todi et al . , 2010 ) . This is supported by the observation that Ubp2 is among the largest yeast DUBs . In addition , several residues of Ubp2 were found to be phosphorylated ( Swaney et al . , 2013 ) , suggesting that coordinated ubiquitylation/phosphorylation events could increase its activity . Finally , given that many DUBs often act as part of protein complexes , Ubp2 ubiquitylation could favor its interaction with Ubp12 and/or Cdc48 . This could release autoinhibition by a conformational change , as observed for the DUB Ubp6 upon binding to the proteasome , i . e . a AAA+ ATPase like Cdc48 ( Hanna et al . , 2006 ) . In fact , Cdc48 has been shown to associate with several DUBs ( Ossareh-Nazari et al . , 2010; Papadopoulos et al . , 2017; Rumpf and Jentsch , 2006; Uchiyama et al . , 2002 ) but also recognizes ubiquitylated proteins , consistent with its interaction with both Ubp12 and Ubp2 . Therefore , DUB ubiquitylation could allow recruitment of Cdc48 and provide a platform guiding DUBs to their relevant substrates . This would also justify the need for Fzo1-Cdc48 physical interaction . In fact , a local regulation of Fzo1 by Cdc48 could allow increased efficiency of the Cdc48-DUB cascade on Fzo1 regulation . In conclusion , our results suggest that Cdc48 serves as a binding platform allowing cross-talk regulation between DUBs , bringing new insights into the knowledge of ubiquitin biology . These general findings open new perspectives to address some poorly understood questions , e . g . how Cdc48 regulates homotypic fusion events and how DUBs are interdependently regulated , possibly accounting for the multitude of DUBs present in a cell .
See Table 1 for details of all yeast strains used . Except for Δpdr5 Δsnq2 ( YGA58 , from J . Dohmen ) and ufd1-2 , npl4-1 and their corresponding wild type ( DF5 , from S . Jentsch ) all other yeast strains are isogenic to the S288c ( Euroscarf ) . They were grown according to standard procedures to the exponential growth phase at 30°C ( unless stated otherwise ) on complete ( YP ) or synthetic ( SC ) media supplemented with 2% ( w/v ) glucose ( D ) , 2% ( w/v ) galactose or 2% ( w/v ) lactate ( Lac ) . Cycloheximide ( CHX ) ( Sigma , Germany ) ( 100 µg/ml for protein shut-down , or 0 . 5 μg/ml when indicated , from a stock of 10 mg/ml in H2O ) or MG132 ( Calbiochem ) ( 50 or 100 μM from a stock of 10 mM in DMSO ) was added when indicated . All plasmids used in this study are described in Table 2 . Plasmid #65 , encoding a non-tagged Ubp12C372S variant , expressed under the control of the ADH1 promoter , was amplified from Ubp12C372S-Flag and cloned with Pst1 , Sal1 into the same sites of YEplac195 . Plasmid #150 , encoding Cdc48A547T was generated by point mutagenesis using plasmid #75 . All antibodies used in this study are described in Table 3 . For growth assays , serial 1:5 dilutions of exponentially growing cells using a starting OD600 of 0 . 5 or 0 . 005 were spotted on YP or SC media containing glucose or lactate and were grown at 30°C or 37°C , as indicated . For analysis of protein steady state levels , total proteins from 3 OD600 exponentially growing cells were extracted at alkaline pH ( Escobar-Henriques et al . , 2006 ) and analyzed by SDS-PAGE and immunoblotting . To monitor protein turnover , cycloheximide ( 100 µg/ml ) was added to exponential cells . Samples of 3 OD600 cells were collected at the indicated time points and total proteins were extracted and analyzed as described above . For monitoring proteasome-dependent degradation of endogenous Fzo1 in wt and cdc48-2 cells , additionally deleted for SNQ2 and PDR5 , YPD media was used ( Liu et al . , 2007 ) , and cells were treated with 50 μM MG132 , 30 min before adding cycloheximide . For monitoring proteasome-dependent degradation of Ubp2 , expressed from plasmid #59 , SCD media was used , and 50 μM MG132 was added 1 hr before starting the cycloheximide chase . Western blots were quantified using Image Quant ( GE Healthcare , Illinois , USA ) . Levels of the protein of interest at time zero were set to 1 . Mean values are shown and the error bars reflect the standard deviation ( SD ) . Total proteins were extracted as described above for the analysis of protein steady state levels but solubilized in LDS buffer ( Thermo Fisher Scientific , Massachusetts , USA ) . Samples were run on precast 4–12% bis-tris gels ( Thermo Fisher Scientific ) using MES buffer ( 50 mM MES , 50 mM Tris Base , 0 . 1% SDS , 1 mM EDTA , pH 7 . 3 ) and transferred to PVDF membranes . Membranes were treated with denaturing solution ( 6 M guanidium chloride , 20 mM Tris pH 7 . 5 , 1 mM PMSF , 5 mM β-mercaptoethanol ) for 30 min and then washed before blocking . Proteins were detected with a ubiquitin-specific antibody ( P4D1; Cell Signaling , Massachusetts ) and a Tpi1-specific antibody , as a loading control . Quantifications were performed using Image Quant ( GE Healthcare ) . Wt values were set to one and the mutants are shown in relation to the wt . Mean values are shown and the error bars reflect the standard deviation ( SD ) . Immunoprecipitation of Ubp12C372S-Flag was performed as follows: 160 OD600 of yeast cells grown in SCD media to the exponential growth phase were disrupted with glass beads ( 0 . 4–0 . 6 µm ) in TBS . After centrifugation , at 16000 g for 10 min , the supernatant was employed to perform an overnight precipitation of Ubp12C372S-Flag , using Flag-coupled beads ( Sigma-Aldrich ) . Elution was performed for 2 hr shaking at 4°C with the 3xFlag-peptide ( Sigma; 200 µg/ml final concentration ) in the following buffer: 50 mM Tris-HCl pH 7 . 5 , 50 mM NaCl . After adding Laemmli buffer , the eluate was split in two , proteins were then resolved in 7% Tris-acetate gels as described ( Cubillos-Rojas et al . , 2012 ) . After transfer , the nitrocellulose membrane was divided in two: one half was immunoblotted with a Flag-specific ( Sigma ) and the other half with a ubiquitin-specific antibody ( P4D1; Cell Signaling ) . Immunoprecipitation of Ubp2C745S-Flag was performed as follows: 160 OD600 of yeast cells grown in SCD media to the exponential growth phase were disrupted with glass beads ( 0 . 4–0 . 6 µm ) in RIPA buffer without detergents ( HEPES-KOH 40 mM pH 7 . 6 , NaCl 150 mM , EDTA 5 mM ) . After centrifugating at 16000 g for 10 min , the supernatant was diluted in an equal volume of RIPA buffer containing 2X detergents , so that the final composition was HEPES-KOH 40 mM pH 7 . 6 , NaCl 150 mM , EDTA 5 mM , Triton X100 1% , SDS 0 . 1% , sodium deoxycholate 0 . 5% . After sonication for 15 min at 4°C in a water bath , denatured cytosolic fractions were employed to precipitate Ubp2C745S-Flag . Flag-coupled beads ( Sigma-Aldrich ) were used for overnight immunoprecipitation and protein elution was performed with Laemmli buffer for 20 min shaking at 40°C . The eluate was split in two and resolved in 8% Tris-glycine gels . After transfer , the nitrocellulose membrane was divided in two: one half of the eluate was immunoblotted with a Flag-specific ( Sigma ) and the other half with a ubiquitin-specific antibody ( P4D1; Cell Signaling ) . Fzo1 ubiquitylation was analyzed as follows: 160 OD600 cell pellets of exponentially growing cultures were used to obtain crude mitochondrial extracts as described ( Anton et al . , 2013 ) . After solubilization with 0 . 2 % NG310 ( Lauryl Maltose Neopentyl Glycol; Anatrace ) for 1 hr rotating at 4°C , samples were centrifuged and 10% of the supernatant was kept as input material . After denaturing in Laemmli buffer for 20 min shaking at 40°C samples were resolved by SDS-PAGE . If necessary , the remaining 90% of the supernatant was incubated with HA-coupled beads ( Sigma-Aldrich ) overnight rotating at 4°C . Three washes were performed with 0 . 2 % NG310 in TBS . HA-Fzo1 was eluted in 50 µl of Laemmli buffer for 20 min shaking at 40°C and analyzed by SDS-PAGE . Proteins were transferred onto nitrocellulose membranes and subsequently immunoblotted using an HA-specific antibody ( Roche , Switzerland ) . Yeast strains were transformed with mitochondrial-targeted GFP , grown on YPD or SC media to the exponential phase and analyzed as described ( Escobar-Henriques et al . , 2006 ) by epifluorescence microscopy ( Axioplan 2; Carl Zeiss MicroImaging , Inc . , Germany ) using a 100x oil-immersion objective . Images were acquired with a camera ( AxioCam MRm , Carl Zeiss MicroImaging , Inc . ) and processed with Axiovision 4 . 7 ( Carl Zeiss MicroImaging , Inc . ) . RNA was isolated from 2 OD600 exponentially growing yeast cells using the NucleoSpin RNA kit ( Macherey Nagel , Germany ) . cDNA was synthesized using the SuperScript III First-Strand Synthesis System ( Invitrogen , Massachusetts , USA ) . mtDNA was quantified by the amplification of COX3 and normalized to ACT1 ( as housekeeping gene ) . Essentially , a dilution of 1:100 of the cDNA was used for the amplification of COX3 ( fw: TTGAAGCTGTACAACCTACC; rv: CCTGCGATTAAGGCATGATG ) and ACT1 ( fw: CACCCTGTTCTTTTGACTGA; rv: CGTAGAAGGCTGGAACGTTG ) by RT-PCR using the Power SYBR Green Master Mix ( AppliedBioSystems ) and three technical replicates for each of the six biological replicates . The ∆CT was calculated using the Livak/2-∆∆CT method ( Livak and Schmittgen , 2001 ) and the fold change of COX3 RNA content in ∆fzo1 and cdc48-2 was calculated in relation to wt . In vitro deubiquitylation assays were performed as described ( Hospenthal et al . , 2015 ) , Essentially , purified K48 or K63 multi-Ub ( BostonBiochem ) or di-Ub chains ( kindly gifted by Thomas Hermanns ) were treated with the DUBs USP2 ( BostonBiochem ) , USP21 ( kindly gifted by Selver Altin ) or Ubp12 . Ubp12 was purified as described above , for the analysis of Ubp12 ubiquitylation , but glycerol to the final concentration of 10% was added , instead of Laemmli . Aliquots of 18 µl , corresponding to 80 OD600 yeast cells , were frozen in liquid nitrogen and stocked at −80°C until further use . For the DUB assay , per reaction , one aliquot of purified Ubp12-flag , 3 µM USP2 or 5 µM USP21 were pre-incubated with 1x DUB dilution buffer ( 25 mM Tris pH 7 . 5 , 10 mM DTT , 150 mM NaCl ) for 10 min at RT . After pre-incubation , the DUBs were mixed with di- or multi-Ub chains to a final concentration of 5 µM in 1x DUB buffer ( 10x DUB buffer: 500 mMTris pH 7 . 5 , 500 mMNaCl , 50 mM DTT ) . Different incubation conditions were used: Ubp12 was incubated with the Ub chains for 45 min at 30°C , USP2 and USP21 for 30 min at 37°C . The reactions were stopped by adding 4x Laemmli buffer . These mixtures were incubated for 20 min at 40°C shaking and further run on an 11% Tris-Tricine SDS-PAGE and transferred onto a PVDF membrane . Ponceau S was used to stain the membrane and after destaining with methanol for 5 min , the membrane was incubated in denaturing solution ( 6M guanidium chloride , 20 mMTris pH 7 . 5 , 1 mM PMSF , 5 mMβ-mercaptoethanol ) for 30 min . Extensive washing was done in TBS-T before blocking the membrane over night with 5% milk in TBS . Results were analyzed by immunoblotting using a Ub-specific antibody .
Cdc48/p97 , a ubiquitin-selective chaperone , orchestrates the function of E3 ligases and deubiquitylases ( DUBs ) . Here , we identify a new function of Cdc48 in ubiquitin-dependent regulation of mitochondrial dynamics . The DUBs Ubp12 and Ubp2 exert opposing effects on mitochondrial fusion and cleave different ubiquitin chains on the mitofusin Fzo1 . We demonstrate that Cdc48 integrates the activities of these two DUBs , which are themselves ubiquitylated . First , Cdc48 promotes proteolysis of Ubp12 , stabilizing pro-fusion ubiquitylation on Fzo1 . Second , loss of Ubp12 stabilizes Ubp2 and thereby facilitates removal of ubiquitin chains on Fzo1 inhibiting fusion . Thus , Cdc48 synergistically regulates the ubiquitylation status of Fzo1 , allowing to control the balance between activation or repression of mitochondrial fusion . In conclusion , we unravel a new cascade of ubiquitylation events , comprising Cdc48 and two DUBs , fine-tuning the fusogenic activity of Fzo1 .
Mitochondria are little compartments within a cell that produce the energy needed for most biological processes . Each cell possesses several mitochondria , which can fuse together and then break again into smaller units . This fusion process is essential for cellular health . Two proteins in the cell have a major role in controlling mitochondrial fusion: Ubp12 and Ubp2 . Ubp12 prevents fusion , while Ubp2 activates it . These molecules carry out their roles by acting on a third protein called mitofusin , which is a key gatekeeper of the fusion mechanism . Cells often ‘tag’ proteins with small molecules called ubiquitin to change the protein’s role and how it interacts with other cellular structures . Depending on how they are ‘tagged’ , mitofusins can exist in two forms . One type of tagging means that the protein then promotes fusion of the mitochondria; the other leads to the mitofusin being destroyed by the cell . It is still unclear how Ubp12 , Ubp2 and the different forms of mitofusins interact with each other to finely control mitochondrial fusion . Here , Simões , Schuster et al . clarify these interactions in yeast and show how these proteins are themselves regulated . Ubp2 promotes fusion by attaching to the mitofusin that is labeled to be destroyed , and removing this tag: the mitofusin will then not be degraded , and can promote fusion . Ubp12 prevents fusion through two mechanisms . First , it can remove the ‘pro-fusion’ tag on the mitofusin that prompts mitochondrial fusion . Second , Simões , Schuster et al . now show that Ubp12 also inhibits Ubp2 and its fusion-promoting activity . In turn , the experiments reveal that a master protein called Cdc48 can control the entire Ubp12-Ubp2-mitofusin pathway . Cdc48 directly represses Ubp12 and therefore its anti-fusion activity . This inhibition also leaves Ubp2 free to stimulate fusion through its action on mitofusin . The molecules involved in controlling mitochondrial fusion in yeast are very similar to the ones in people . In humans , improper regulation of mitofusins causes an incurable disease of the nerves and the brain called Charcot-Marie-Tooth 2A . Understanding how the fusion of mitochondria is controlled can lead to new drug discoveries .