A protein quality control pathway at the mitochondrial outer membrane
Proper protein folding is essential for organelle and cell homeostasis . Proteins may fail to achieve or retain their functional conformations due to genetic or environmental insults and cells have evolved elaborate protein quality control ( QC ) mechanisms to combat misfolding . QC systems recognize and refold misfolded proteins and , when not possible , sequester or degrade them to prevent their often deleterious accumulation ( Kevei et al . , 2017 ) . Such degradation frequently occurs via the ubiquitin-proteasome system ( UPS ) , where substrates are modified with ubiquitin leading to their destruction by the 26S proteasome ( Metzger et al . , 2012 ) . Ubiquitin modification requires the action of ubiquitin-activating enzyme ( E1 ) , followed by either sequential or concerted activity of ubiquitin-conjugating enzymes ( E2 ) and substrate-specific ubiquitin ligases ( E3 ) . Multiple rounds of ubiquitination result in polyubiquitin chains , which can serve as targeting signals for recognition and degradation by 26S proteasomes ( Akutsu et al . , 2016; Thrower et al . , 2000 ) . Other classes of factors play critical roles in substrate recognition and in their extraction from associated proteins or membranes post-ubiquitination , as well as in shuttling to proteasomes ( Buchberger et al . , 2015; Ye et al . , 2017; Zientara-Rytter and Subramani , 2019 ) . Distinct , yet overlapping UPS machinery found at different subcellular locations mediates localized QC of misfolded proteins ( Sontag et al . , 2017 ) . The degradation of misfolded proteins at the ribosome , endoplasmic reticulum ( ER ) , nucleus , inner nuclear envelope , and cytosol is mediated by dedicated UPS machinery ( Boban and Foisner , 2016; Brandman and Hegde , 2016; Comyn et al . , 2014; Gamerdinger , 2016; Jones and Gardner , 2016; Zattas and Hochstrasser , 2015 ) . Much of what is known about these subcellular QC pathways was uncovered in yeast using model misfolded substrates ( Bays et al . , 2001; Gardner et al . , 2005; Huyer et al . , 2004; Stolz and Wolf , 2012; Vashist and Ng , 2004 ) and has served as the basis for characterizing mammalian degradation pathways . The most extensively studied organelle-based QC pathway is ER-associated degradation ( ERAD ) . Early steps in ERAD pathways , such as substrate recognition and ubiquitination , are distinct for different substrates and defined ( at least in yeast ) by the location of the misfolded domain ( Carvalho et al . , 2006; Huyer et al . , 2004; Preston and Brodsky , 2017; Ruggiano et al . , 2014 ) . Generally , pathways converge post-ubiquitination , where the Cdc48-Npl4-Ufd1 AAA-ATPase complex and its associated co-factors facilitate unfolding and/or extraction of substrates from their natural environments prior to targeting to 26S proteasomes for degradation ( Olszewski et al . , 2019; Wolf and Stolz , 2012 ) . Mitochondrial proteins are subject to ongoing oxidative insults that can result in damage , misfolding , and dysfunction . Mitochondria require mechanisms to eliminate these proteins to maintain organellar integrity and essential functions ( Voos et al . , 2016 ) . In mammalian cells , the well-characterized Parkin and PINK1 ubiquitin-dependent mitophagy pathway removes portions of , or entire , damaged mitochondria ( McWilliams and Muqit , 2017; Pickles et al . , 2018 ) . In yeast , however , mitophagy appears to function primarily to adapt to metabolic changes , rather than in protein QC ( Fukuda and Kanki , 2018; Kanki et al . , 2015 ) . Furthermore , there is little evidence indicating that mitophagy is ubiquitin-dependent , in accordance with the absence of obvious yeast orthologues of Parkin or PINK1 ( Belgareh-Touzé et al . , 2017; Tan et al . , 2016 ) . In contrast to mitophagy , mitochondria-associated degradation ( MAD ) pathways for individual misfolded or damaged mitochondrial proteins are not as well-established in mammals or yeast . Proteases resident to the mitochondrial matrix , inner membrane ( IM ) , and intermembrane space ( IMS ) can act on damaged or aggregated proteins in these compartments ( Bohovych et al . , 2015 ) . While there is no evidence for proteasomes inside mitochondria , the mitochondrial outer membrane ( MOM ) is fully accessible to cytosolic proteasomes . In fact , the UPS is known to play a critical role in mitochondrial morphology , dynamics , inheritance , and in the degradation of import-deficient mitochondrial IMS proteins and mitochondria-mislocalized tail-anchored proteins ( Altmann and Westermann , 2005; Bragoszewski et al . , 2017; Cohen et al . , 2008; Fisk and Yaffe , 1999; Goodrum et al . , 2019; Matsumoto et al . , 2019; Rinaldi et al . , 2008 ) . A limited number of MOM proteins have also been identified as specific UPS targets in yeast: Fzo1 , Mdm12 , Mdm34 , Msp1 , and Tom70 ( Belgareh-Touzé et al . , 2017; Cohen et al . , 2008; Fritz et al . , 2003; Ota et al . , 2008; Wu et al . , 2016 ) . All of these are native ( i . e . non-misfolded ) proteins , whose ubiquitination and/or degradation may be critical for homeostasis and not obviously related to QC . Of these , only Fzo1 has been examined in detail . The regulated recognition and ubiquitination of this mitofusin by SCFMdm30 ( Skp1-Cullin-F-box E3 with F-box protein Mdm30 ) and its subsequent proteasomal degradation are integral to the process of MOM fusion ( Cohen et al . , 2011; Cohen et al . , 2008; Escobar-Henriques et al . , 2006 ) . However , for the few other MOM proteins where ubiquitination has been analyzed , the E3 Rsp5 has been implicated ( Belgareh-Touzé et al . , 2017; Goodrum et al . , 2019; Kowalski et al . , 2018; Wu et al . , 2016 ) . The involvement of Cdc48 co-factors , Vms1 and Doa1 , has also been both reported and disputed for particular MOM proteins ( Chowdhury et al . , 2018; Esaki and Ogura , 2012; Heo et al . , 2010; Wu et al . , 2016 ) . The degradation of tail-anchored proteins mislocalized to the MOM uniquely requires the AAA-ATPase Msp1 for extraction from the MOM prior to transfer to the ER where they are degraded by ERAD machinery ( Matsumoto et al . , 2019; Okreglak and Walter , 2014; Wohlever et al . , 2017 ) . Thus , a universal MAD pathway for MOM proteins has not been described and many steps in substrate degradation remain unexamined . Similarly , UPS components acting on misfolded MOM proteins have yet to be investigated . In this study , we establish temperature-sensitive ( ts- ) peripheral MOM proteins ( sam35-2HAts and sen2-1HAts ) as QC substrates . The ts- nature of these substrates , coupled with their tight association with the MOM , enabled study of their mitochondrial degradation without concerns of mislocalization to the cytosol or elsewhere . We utilize these substrates to define a MAD pathway for non-native proteins . The proteasomal degradation of these MAD QC substrates occurs at the MOM and requires specific cytosolic and mitochondrial UPS components , most of which are conserved in higher eukaryotes . Our results reveal a requirement for factors not previously implicated in the degradation of native MAD substrates and the combination of components identified defines a distinct QC pathway .
Our knowledge of protein QC in the ER , cytosol , and nucleus derives in part from the study of proteins that undergo temperature-dependent misfolding and degradation ( Biederer et al . , 1996; Gardner et al . , 2005; Khosrow-Khavar et al . , 2012; Ravid et al . , 2006; Wang and Prelich , 2009 ) . To elucidate how misfolded mitochondrial proteins are targeted for destruction , we exploited two previously-identified yeast ts- alleles , sam35-2 and sen2-1 ( Li et al . , 2011; Milenkovic et al . , 2004; Winey and Culbertson , 1988 ) , whose degradation has not been assessed . Sam35 and Sen2 are essential MOM proteins . Sam35 is the substrate receptor of the MOM-embedded multiprotein sorting and assembly machinery ( SAM ) complex required for assembly of β-barrel proteins into the MOM ( Chan and Lithgow , 2008; Kozjak et al . , 2003; Kutik et al . , 2008; Milenkovic et al . , 2004 ) . Although Sam35 contains no apparent membrane spans , it is tightly embedded at the MOM via the Sam50 protein ( Kutik et al . , 2008 ) . Sen2 provides endonuclease activity for the multi-subunit tRNA splicing endonuclease complex and also cleaves a mitochondria-targeted non-stop mRNA ( Ho et al . , 1990; Tsuboi et al . , 2015; Winey and Culbertson , 1988 ) . The tRNA splicing complex resides on the MOM in yeast , with Sen2 potentially anchoring it to the membrane via a hydrophobic segment ( Trotta et al . , 1997; Yoshihisa et al . , 2003 ) . We first determined that sam35-2ts and sen2-1ts encoded full-length proteins . Each contained multiple missense mutations ( Figure 1—figure supplement 1A ) that likely account for the phenotypes reported ( Li et al . , 2011; Milenkovic et al . , 2004; Winey and Culbertson , 1988 ) . To facilitate detection , sequence encoding an HA tag was added to the C-terminus of each to generate sam35-2HAts and sen2-1HAts . Genome-integrated versions of sam35-2HAts and sen2-1HAts support cell viability at the permissive ( 25°C ) but not non-permissive temperature ( 37°C; Figure 1A ) , consistent with the previously-described untagged versions ( Li et al . , 2011; Milenkovic et al . , 2004; Winey and Culbertson , 1988 ) . To determine whether their ts- phenotypes are indicative of protein misfolding that may lead to instability , we assessed sam35-2HAts and sen2-1HAts turnover by cycloheximide ( CHX ) chase when expressed from centromeric ( CEN ) yeast plasmids . Cells were grown at 25°C until the addition of CHX at time ‘zero’ , when the temperature was either maintained at 25°C or raised to 37°C . The addition of CHX prior to raising the temperature minimizes the contribution of newly synthesized ts- proteins that may misfold prior to reaching the mitochondria . We find that increasing the temperature to 37°C results in dramatic destabilization of the ts- proteins relative to the WT proteins ( Figure 1B ) . Chromosomal versions behaved similarly ( Figure 1—figure supplement 1B ) . Thus , the ts- nature of these alleles is likely attributable to misfolding leading to destabilization at the non-permissive temperature . Next , agarose-embedded cells expressing GFP-tagged sam35-2ts and sen2-1ts were used to assess whether these non-transmembrane MOM proteins remain mitochondrially-associated following the increase to the non-permissive temperature . For this , the temperature was raised to 37°C for 5 min ( sen2-1GFPts ) or 15 min ( sam35-2GFPts ) , times at which sam35-2GFPts and sen2-1GFPts were already destabilized by CHX chase ( Figure 1—figure supplement 1C ) . Co-expression of sam35-2GFPts and sen2-1GFPts with a mitochondrial marker ( mtRFP ) demonstrated that the ts- proteins remain mitochondrial after the temperature increase ( Figure 1C ) . To bypass difficulties associated with imaging these low abundance ts- proteins , we isolated mitochondria by subcellular fractionation and assessed the localization of destabilized sam35-2HAts and sen2-1HAts . Using the same conditions as for microscopy , we found that the HA-tagged ts- proteins are destabilized upon the addition of CHX ( Figure 1—figure supplement 1D ) and that protein turnover was still evident in the spheroplasted yeast used for fractionation ( WT; Figure 1—figure supplement 1E ) . Consistent with our microscopy , sam35-2HAts and sen2-1HAts fractionated almost exclusively to the mitochondrial pellet at 37°C ( P; Figure 1D ) . A similar fractionation pattern was observed with chromosomal HA-tagged ts- alleles ( Figure 1—figure supplement 1F ) . Therefore , sam35-2HAts and sen2-1HAts are destabilized at the non-permissive temperature yet remain mitochondrially-localized , validating their use as model MAD QC substrates . Since proteasomes have broad roles in mitochondrial homeostasis and morphology , we assessed whether the degradation of these MOM ts- proteins was dependent on proteasome function . The turnover of sam35-2HAts and sen2-1HAts was assessed at the non-permissive temperature in strains containing conditional mutations in the 20S core ( pre1-1 pre2-2; Figure 2A ) or the 19S cap ( cim3-1; Figure 2B ) of the 26S proteasome . Both ts- proteins were substantially stabilized in these mutants relative to isogenic WT strains . In contrast , vacuolar or mitochondrial resident proteases were not required for sam35-2HAts or sen2-1HAts turnover ( Figure 2—figure supplement 1A and B ) . After establishing that the stabilization of the ts- proteins in pre1-1 pre2-2 spheroplasts mirrors that observed in intact cells ( Figure 1—figure supplement 1E ) , we assessed whether sam35-2HAts and sen2-1HAts remain mitochondrial when proteasome function is impaired . Even after 40 min at the non-permissive temperature , the majority of both sam35-2HAts and sen2-1HAts accumulated in the 12 , 000xg mitochondrial pellet ( Figure 2C ) . Extraction of both proteins from this pellet fraction by sodium carbonate excluded the possibility that this represents aggregated protein ( Figure 2—figure supplement 1C ) . This crude mitochondrial pellet also contains ~20% of total ER as assessed by measuring levels of the integral ER membrane protein Cue1 ( Figure 2—figure supplement 1D ) . For this reason , we wished to exclude the possibility that these substrates were being translocated to the ER for degradation . Such a pathway has recently been described for tail-anchored proteins that are mislocalized to mitochondria ( Matsumoto et al . , 2019 ) . Further purification of the crude 12 , 000xg mitochondrial pellet isolated from pre1-1 pre2-2 cells via sucrose gradient ultracentrifugation removes ~90% of contaminating ER , leaving only ~2% of total ER in these purified mitochondria ( Cue1; Figure 2—figure supplement 1E ) . On the other hand , this purified mitochondrial fraction retains ~70% of the ts- proteins . As peripheral membrane proteins could dissociate from mitochondria during manipulation , 70% may be an underrepresentation of the mitochondria-associated pool in vivo . Thus , these data indicate that sam35-2HAts and sen2-1HAts primarily accumulate at the mitochondria when their proteasomal degradation is blocked . Consistent with being UPS substrates , stabilization of both sam35-2HAts and sen2-1HAts was observed in a ts- mutant of the ubiquitin-activating enzyme ( uba1-204; Figure 2D; Ghaboosi and Deshaies , 2007 ) , indicating that ubiquitination is required for their degradation . Moreover , ubiquitin-modified forms of sam35-2HAts and sen2-1HAts were evident when proteasome function was inhibited ( Figure 2E and F and see Figure 2—figure supplement 1F for an additional control ) , as indicated by characteristic higher molecular weight bands and a smear representing highly ubiquitinated species ( Emmerich and Cohen , 2015 ) . By comparison , ubiquitinated forms of WT SAM35HA and SEN2HA are much less prevalent ( Figure 2—figure supplement 1G ) . We further assessed whether ubiquitin-modified forms of the ts- proteins were localized to mitochondria under conditions of proteasome impairment . Ubiquitinated sam35-2HAts and sen2-1HAts both accumulated largely in the mitochondrial pellet fraction ( P12 , 000xg; Figure 2—figure supplement 1H ) and to a lesser extent in the soluble fraction , correlating with the relative distributions of unmodified species . Taken together , these data establish roles for ubiquitination and proteasomal degradation in the turnover of sam35-2HAts and sen2-1HAts at the MOM . To date , the only ubiquitin ligases known to act on mitochondrial proteins in yeast are SCFMdm30 and Rsp5 ( Belgareh-Touzé et al . , 2017; Cohen et al . , 2008; Escobar-Henriques et al . , 2006; Kowalski et al . , 2018; Wu et al . , 2016 ) . However , the known mitochondrial substrates for these E3s are native proteins . We assessed whether these E3s also play a role in MAD of QC substrates and found that strains lacking either Mdm30 or Rsp5 function did not affect sam35-2HAts or sen2-1HAts stability ( Figure 3—figure supplement 1A and B ) . To identify factors involved in targeting these ts- MAD substrates , we screened a yeast deletion library consisting of non-essential known or putative UPS components ( Ravid and Hochstrasser , 2007 ) by CHX chase . Interestingly , we found that san1Δ impaired turnover of sam35-2HAts ( Figure 3A , middle panel ) , while ubr1Δ impaired turnover of sen2-1HAts ( Figure 3B , middle panel ) . San1 has been implicated in QC of cytosolic and nuclear proteins ( Gardner et al . , 2005; Heck et al . , 2010 ) . Ubr1 is the E3 that ubiquitinates N-end rule substrates , but also plays a prominent role in the turnover of cytosolic proteins and , in some instances , may also degrade ER proteins ( Bartel et al . , 1990; Comyn et al . , 2016; Eisele and Wolf , 2008; Heck et al . , 2010; Khosrow-Khavar et al . , 2012; Rao et al . , 2001; Scazzari et al . , 2015; Stolz et al . , 2013; Summers et al . , 2013 ) . Since there are examples where these two E3s functionally interact , we assessed turnover in a san1Δ ubr1Δ double deletion strain . While ubr1Δ alone did not affect sam35-2HAts turnover ( Figure 3—figure supplement 1C ) , loss of Ubr1 in conjunction with san1Δ led to greater stabilization than loss of San1 alone ( Figure 3A , lower panel ) , indicating that Ubr1 can target sam35-2HAts in the absence of San1 . Loss of San1 , alone or in conjunction with ubr1Δ , did not affect the stability of sen2-1HAts ( Figure 3—figure supplement 1D and Figure 3B , lower panel ) . Notably , the polyubiquitination of sam35-2HAts ( Figure 3C ) and sen2-1HAts ( Figure 3D ) was greatly reduced when San1 or Ubr1 , respectively , were deleted . Neither ts- protein was entirely stabilized by san1Δ ubr1Δ , suggesting that other E3s , perhaps with overlapping specificity or essential functions , may also act on these substrates . Likewise , loss of any single E2 did not stabilize sam35-2HAts ( data not shown ) . Loss of Ubc4 , an E2 known to function with Ubr1 for select substrates , stabilized sen2-1HAts to a similar degree as ubr1Δ ( Figure 3E ) . To confirm that Ubr1 and San1 can act on ts- proteins at the MOM , we examined the localization of sam35-2HAts and sen2-1HAts by fractionation in san1Δ and ubr1Δ , respectively . In these strains , both ts- proteins still accumulated almost exclusively in the pellet fraction ( Figure 3F and G ) . These results establish that San1 and Ubr1 can function as components of the MAD machinery for proteasome-mediated degradation of sam35-2HAts and sen2-1HAts , respectively . The cytosolic SSA family of Hsp70 chaperones ( Ssa1 , Ssa2 , Ssa3 , and Ssa4 ) is broadly involved in protein folding and refolding , but also plays a role in the degradation of several San1 and Ubr1 QC substrates ( Guerriero et al . , 2013; Heck et al . , 2010; Prasad et al . , 2010; Stolz et al . , 2013 ) . During protein QC , these chaperones appear to prevent protein aggregation and facilitate ubiquitination ( Huyer et al . , 2004; Metzger et al . , 2008; O'Donnell et al . , 2017; Park et al . , 2007; Scazzari et al . , 2015; Stolz et al . , 2013 ) . However , they have not been implicated in MAD . Interestingly , both sam35-2HAts and sen2-1HAts were strongly stabilized in a conditional yeast strain where three SSA family members are deleted and the fourth ( SSA1 ) is ts- ( ssa1-45ts; ssa1-45ts ssa2Δ ssa3Δ ssa4Δ ) relative to the control strain ( SSA1; SSA1 ssa2Δ ssa3Δ ssa4Δ; Figure 4A ) . SSA Hsp70 chaperones can play a role in the sorting of mitochondrial proteins to and into mitochondria ( Deshaies et al . , 1988; Eliyahu et al . , 2012; Young et al . , 2003 ) . To rule out the possibility that the stabilization of mitochondrial ts- alleles in the ssa1-45ts strain is reflective of a defect in mitochondrial targeting , mitochondrial fractionation was performed . Both sam35-2HAts and sen2-1HAts were localized to the pellet fraction ( Figure 4B ) , where they were extractable by sodium carbonate ( Figure 4—figure supplement 1A ) . Notably , the SSA1 chaperone was also required for ubiquitination: despite the accumulation of unmodified sam35-2HAts and sen2-1HAts , ubiquitin-modified forms are dramatically reduced when SSA1 is inactivated ( Figure 4C ) . This is consistent with the established QC function for SSA chaperones in engaging substrates prior to their ubiquitination ( Shiber and Ravid , 2014 ) . In contrast to the ts- proteins , degradation of the native MOM UPS substrate , Fzo1HA , was largely unaffected by loss SSA function ( Figure 4—figure supplement 1B ) . This is consistent with a dichotomy in chaperone requirements between native and non-native MAD substrates . Hsp40 co-chaperones ( J-proteins ) stimulate the ATPase activity of Hsp70 , which is required for substrate interactions . In particular , Sis1 and Ydj1 play roles in SSA-dependent protein QC throughout the cell ( Heck et al . , 2010; Lu and Cyr , 1998; Prasad et al . , 2018; Shiber and Ravid , 2014; Summers et al . , 2013 ) . Depletion of ~90% of Sis1 protein using Sis1 DAmP cells ( Figure 4—figure supplement 1C ) slowed the turnover of both sam35-2HAts and sen2-1HAts ( Figure 4D ) , while loss of Ydj1 or its ortholog , Hlj1 , did not ( Figure 4—figure supplement 1D ) . Recent studies indicate that the degradation of many San1 and Ubr1 cytosolic substrates occurs following nuclear import that is dependent both on Ydj1 and the Hsp70 nucleotide exchange factor ( Hsp110 ) Sse1 ( Prasad et al . , 2018; Samant et al . , 2018 ) . We did not detect a role for Sse1 in the degradation of sam35-2HAts and sen2-1HAts ( Figure 4—figure supplement 1E ) . Loss of other factors implicated in San1 and Ubr1 nuclear import-dependent QC , including Hsc82/Hsp82 , Sti1 , Hsp104 , and Dsk2 were also without effect on the degradation of sam35-2HAts and sen2-1HAts ( Figure 4—figure supplement 1F ) . These results establish that the degradation of the mitochondrial QC substrates is dependent on both Hsp70 and Hsp40 chaperones , but independent of nuclear import . The AAA-ATPase Cdc48 plays a broad role in many QC pathways , generally functioning as a protein ‘unfoldase’ or ‘segregase , ’ while also maintaining protein solubility prior to proteasomal degradation ( Neal et al . , 2017; Ye et al . , 2017 ) . Cdc48 and its co-factors Npl4 and Ufd1 have been implicated in the UPS-mediated turnover of several yeast MOM proteins ( Cohen et al . , 2008; Heo et al . , 2010; Wu et al . , 2016 ) . Both sam35-2HAts and sen2-1HAts were stabilized in conditional Cdc48 , Npl4 , and Ufd1 strains ( Figure 5A ) . In contrast , loss of Msp1 , another AAA-ATPase that localizes to mitochondria and dislocates mislocalized tail-anchored proteins from the MOM ( Chen et al . , 2014; Okreglak and Walter , 2014; Wohlever et al . , 2017 ) , stabilized neither sam35-2HAts nor sen2-1HAts ( Figure 5—figure supplement 1A ) . Given the role of the Cdc48-Npl4-Ufd1 complex as a segregase , we examined whether the ts- proteins accumulated at mitochondria when activity of the complex is compromised , as might be predicted . For both cdc48-3 and its isogenic WT parental strain , interpretation of fractionation results was clouded by some baseline protein detected in the post-mitochondrial supernatant ( Figure 5B , left panels ) . For npl4-1 and ufd1-1 , however , both substrates remain primarily mitochondrial ( Figure 5B , middle and right panels ) . This indicates that in the absence of a functional Cdc48-Npl4-Ufd1 complex sam35-2HAts and sen2-1HAts largely retain their mitochondrial localization . The Cdc48-Npl4-Ufd1 complex generally binds to ubiquitinated proteins ( Bodnar et al . , 2018; Park et al . , 2005; Tsuchiya et al . , 2017 ) , acting downstream of E3-mediated ubiquitination . Accordingly , we detected a relative increase in ubiquitinated sam35-2HAts and sen2-1HAts in Cdc48-Npl4-Ufd1 complex mutants ( Figure 5C and Figure 5—figure supplement 1B and C ) . In ERAD , the UBL- and UBA-containing proteins Rad23 and Dsk2 have been implicated as factors that shuttle ubiquitinated substrates from the Cdc48 complex to proteasomes . Deletion of these or another proteasome shuttling factor , Ddi1 , did not affect the degradation of sam35-2HAts or sen2-1HAts ( Figure 5—figure supplement 1D ) , further distinguishing MAD from ERAD . All together , these results indicate that ubiquitinated mitochondrial QC substrates require the Cdc48-Npl4-Ufd1 complex for efficient proteasomal degradation . The Cdc48-Npl4-Ufd1 complex can be recruited to substrates via Cdc48’s interaction with co-factors ( Buchberger et al . , 2001; Buchberger et al . , 2015; Wu et al . , 2016 ) . Vms1 and Doa1 are two cytosolic Cdc48 co-factors implicated in mitochondrial homeostasis ( Heo et al . , 2010; Izawa et al . , 2017; Nielson et al . , 2017; Wu et al . , 2016 ) , although their reported involvement in mitochondrial protein turnover , particularly with respect to Fzo1 , has been inconsistent ( Chowdhury et al . , 2018; Esaki and Ogura , 2012; Wu et al . , 2016 ) . Loss of Vms1 had no effect on the degradation of sam35-2HAts and sen2-1HAts ( Figure 6—figure supplement 1A ) , while loss of Doa1 had a small , but significant effect ( Figure 6A ) . We next examined deletions of each of the UBX ( Ubiquitin-regulatory X ) proteins , a family of Cdc48 binding co-factors containing a C-terminal Ub fold ( UBX ) domain ( Schuberth and Buchberger , 2008; Schuberth et al . , 2004 ) . Notably , only the loss of Ubx2 significantly stabilized sam35-2HAts and sen2-1HAts ( Figure 6B and Figure 6—figure supplement 1B ) . Ubx2 ( as well as its mammalian ortholog , UbxD8 ) is well-characterized as an ER membrane protein with roles in ERAD and lipid droplet homeostasis ( Kolawa et al . , 2013; Neuber et al . , 2005; Schuberth and Buchberger , 2005; Wang and Lee , 2012 ) . We confirmed Ubx2’s role in degradation of the mitochondrial ts- proteins by complementation with FLAG-tagged Ubx2 in the ubx2Δ strain ( Figure 6C ) . Furthermore , redundant functions for Ubx2 and Doa1 were ruled out by a failure of Doa1 overexpression to restore sam35-2HAts or sen2-1HAts degradation in ubx2Δ cells ( Figure 6D ) . As might be predicted given the role of Ubx2 in linking ubiquitinated proteins to the Cdc48 complex , ubiquitinated forms of the ts- substrates accumulated in the absence of Ubx2 ( Figure 6E ) . We also detected a physical association between Ubx2-FLAG and the ts- proteins as assessed by co-immunoprecipitation ( Figure 6F ) . We see no evidence that loss of Ubx2 affects the already slow turnover of either SAM35HA or SEN2HA ( Figure 6—figure supplement 1C ) , and an association between these WT proteins and Ubx2 was also not as pronounced relative to their abundance ( Figure 6—figure supplement 1D ) . However , in agreement with recent reports ( Chowdhury et al . , 2018; Nahar et al . , 2020; Wu et al . , 2016 ) , we find that loss of Ubx2 and , to a lesser extent , Doa1 , stabilizes Fzo1HA , which is a native MOM UPS substrate ( Figure 6—figure supplement 1E and F ) . Importantly , we also establish that Fzo1HA physically interacts with Ubx2 , accompanied by an increase in ubiquitinated forms ( Figure 6—figure supplement 1G and H ) . Our findings are consistent with Ubx2 interacting with UPS-targeted native and misfolded substrates downstream of their ubiquitination to facilitate degradation . Although Ubx2 is an ER transmembrane protein , one study suggested it may also localize to mitochondria ( Wang and Lee , 2012 ) , which was recently corroborated ( Mårtensson et al . , 2019 ) . We also confirmed this by co-localization of Ubx2-GFP with both ER ( Sec63-RFP ) and mitochondrial ( mtRFP ) markers by microscopy ( Figure 6—figure supplement 1I ) . The mitochondrial localization of a portion of Ubx2 was further verified biochemically by isolation of mitochondria largely devoid of co-purifying cytosolic ( PGK ) , ER luminal ( CPY ) , or ER membrane ( Cue1 ) proteins ( Figure 6G ) . As the ER and mitochondria are in close apposition at ER-mitochondrial contact sites , it is possible that ER-localized Ubx2 facilitates mitochondrial protein degradation . Recent studies have also characterized a pathway for degradation of tail-anchored proteins mislocalized to mitochondria that entails Msp1-mediated extraction and subsequent degradation at the ER by ERAD machinery ( Dederer et al . , 2019; Matsumoto et al . , 2019 ) . To exclude a potential role for the ER in sam35-2HAts and sen2-1HAts degradation , we assessed their turnover in mutants of the well-characterized ER-mitochondrial encounter structure ( ERMES ) complex . Loss of individual ERMES subunits reduces ER-mitochondrial tethering by greater than 70% and partially disrupts ion and lipid exchange between the organelles ( Kornmann and Walter , 2010; Lahiri et al . , 2014; Murley and Nunnari , 2016 ) . The turnover of sam35-2HAts and sen2-1HAts was unaffected in strains mutant for each of the four ERMES components ( Figure 6—figure supplement 1J ) , indicating that significant reductions in ER-mitochondrial contact do not impair their degradation . Furthermore , ts- protein degradation is unaffected by combined loss of the major ERAD E2s , Ubc6 and Ubc7 , or E3s , Doa10 and Hrd1 ( Figure 6—figure supplement 1K and L ) , with which Ubx2 functionally and physically interacts at the ER ( Neuber et al . , 2005; Schuberth and Buchberger , 2005 ) . Ubc6 , Ubc7 , and Doa10 have also been implicated in the degradation of mislocalized tail-anchored proteins subsequent to Msp1-dependent removal from mitochondria ( Dederer et al . , 2019; Matsumoto et al . , 2019 ) . Degradation of sam35-2HAts and sen2-1HAtsis independent of Msp1 ( Figure 5—figure supplement 1A ) . Finally , as only ~20% of the ER is found in the mitochondrial pellet fraction at 12 , 000xg ( Figure 2—figure supplement 1D ) , the ts- substrates would be expected to accumulate predominantly in the post-mitochondrial supernatant ( S ) if they were being degraded from the ER . However , fractionation data in the proteasome mutant strain ( Figure 2C and Figure 2—figure supplement 1E ) strongly suggests that ts- protein degradation occurs from the mitochondria . Sam35-2HAts and sen2-1HAts also largely accumulate in the mitochondrial pellet in ubx2Δ cells ( Figure 6H ) . Similar to the ts- proteins , degradation of Fzo1HA , which is a substrate for the cytosolic E3 SCFMDM30 and its cognate E2 Cdc34 ( Cohen et al . , 2008 ) , is unaffected by loss of ERMES components ( Figure 6—figure supplement 1M ) . Fzo1 also accumulates predominantly in the mitochondrial pellet when Ubx2 is absent ( Figure 6—figure supplement 1N ) . Thus , our data indicate that the mitochondrial population of Ubx2 interacts with UPS-targeted MAD substrates post-ubiquitination to facilitate their degradation from the MOM .
Mitochondria are essential to cellular bioenergetics and metabolism . It is therefore vital that QC mechanisms dispose of damaged proteins that can compromise function . In mammalian cells , this is accomplished at the macroscopic level through mitophagy ( Pickles et al . , 2018 ) . Here , we define a UPS-mediated pathway for the degradation of individual dysfunctional yeast MOM proteins using two newly-established model QC substrates , sam35-2HAts and sen2-1HAts ( schematized in Figure 7 ) . While previous mitochondrial UPS substrates have consisted primarily of native proteins ( Belgareh-Touzé et al . , 2017; Cohen et al . , 2008; Heo et al . , 2010; Wu et al . , 2016 ) , sam35-2HAts and sen2-1HAts contain mutations that render them unstable at the non-permissive temperature , and thus mimic damaged , misfolded proteins . These new mitochondrial model substrates have revealed a tightly-coupled degradation pathway at the MOM that requires both cytosolic and mitochondrial machinery . This MAD QC pathway utilizes factors that have not been previously linked to mitochondria or implicated in MAD of native MOM proteins . It is also not identical to any other cellular QC pathway , although there are points of intersection . All of these degradation pathways , unsurprisingly , require 26S proteasome activity . In many cases , the involvement of the Cdc48-Npl4-Ufd1 AAA-ATPase complex represents another point of convergence ( Benischke et al . , 2014; Cohen et al . , 2008; Gallagher et al . , 2014; Heo et al . , 2010; Jarosch et al . , 2002; Tanaka et al . , 2010; Wu et al . , 2016; Xu et al . , 2011 ) . However , tail-anchored proteins mistargeted to mitochondria and unimported mitochondrial precursors that accumulate at the mitochondrial surface following mitochondrial import stress also require the AAA-ATPase Msp1 for their recognition and extraction from mitochondria ( Chen et al . , 2014; Matsumoto et al . , 2019; Okreglak and Walter , 2014; Weidberg and Amon , 2018; Wohlever et al . , 2017 ) . We found Msp1 to be dispensable for the degradation of mitochondrial ts- substrates . It was recently determined that the degradation of these mislocalized tail-anchored proteins involves their re-localization to the ER following Msp1 extraction , where they are then ubiquitinated by ERAD machinery ( Dederer et al . , 2019; Matsumoto et al . , 2019 ) . The MAD QC pathway characterized here is distinct from this pathway: ts- substrates remain mitochondrial and are degraded independently of ERAD E2s or E3s . The molecular determinants , spatial restrictions , and co-factors that underlie ubiquitination and degradation at the mitochondria versus Msp1-dependent ER re-targeting will be of particular interest to elucidate going forward . The Cdc48-Npl4-Ufd1 complex plays a role in both of these pathways and utilizes unique substrate recruitment co-factors . Of these co-factors , Vms1 is recruited to mitochondria from the cytosol in response to translational or oxidative stress ( Izawa et al . , 2017; Nielson et al . , 2017 ) and cytosolic Doa1 is implicated in the turn-over of native MOM proteins ( Wu et al . , 2016 ) . Only loss of Doa1 had an effect on mitochondrial ts- protein degradation . However , we found another Cdc48 complex co-factor , Ubx2 , to have a substantially greater role in degradation of the two ts- substrates . Ubx2 is well-known as an ER transmembrane protein with a role in ERAD . Here , we provide strong evidence for a discrete , functional , mitochondrial pool of Ubx2 . Interestingly , we find that Ubx2 also interacts with Fzo1 at mitochondria and is required for its degradation . This raises the interesting possibility that Ubx2 , as well as its mammalian ortholog UbxD8 , will have a broad role in MAD and mitochondrial homeostasis in addition to its role in ERAD . Consistent with this , while this manuscript was in preparation , a role for Ubx2 in the turnover of mitochondrial precursor proteins arrested in the Tom40 translocon was reported ( Mårtensson et al . , 2019 ) . With regard to ubiquitination , we find no evidence for the involvement of Rsp5 or SCFMdm30 ubiquitin ligases , which are both implicated in ubiquitination of native MOM proteins and/or maintenance of mitochondrial integrity ( Belgareh-Touzé et al . , 2017; Cohen et al . , 2008; Escobar-Henriques et al . , 2006; Fritz et al . , 2003; Wu et al . , 2016 ) . Instead , San1 and Ubr1 , which have broad roles as quality control E3s for misfolded cytosolic and nuclear proteins ( Amm et al . , 2016; Amm and Wolf , 2016; Eisele and Wolf , 2008; Gardner et al . , 2005; Guerriero et al . , 2013; Heck et al . , 2010; Khosrow-Khavar et al . , 2012; Lewis and Pelham , 2009; Nillegoda et al . , 2010; Prasad et al . , 2012; Prasad et al . , 2018; Samant et al . , 2018; Summers et al . , 2013 ) , are required to degrade mitochondrial ts- substrates . Recent studies suggest that the degradation of many cytosolic San1 and Ubr1 substrates requires prior nuclear import ( Prasad et al . , 2018; Samant et al . , 2018 ) . Sam35-2HAts and sen2-1HAts , however , remain mitochondrial when degradation is blocked and specific factors implicated in nuclear import were found to be dispensable . While there have also been reports of Ubr1 contributing to ERAD ( Stolz et al . , 2013 ) , as noted above , MAD QC appears to be distinct from ERAD . For ubiquitination of QC substrates to occur , they must first be recognized as being improperly folded . In most QC systems , this recognition requires chaperones . Here , the involvement of the SSA family of Hsp70s in mitochondrial ts- protein degradation reflects commonality with cytosolic QC pathways and ERAD pathways for cytosolic misfolded domains ( ERAD-C ) . The SSA family of chaperones is required for co-translational folding or import of at least some mitochondrial proteins ( Ben-Menachem et al . , 2018; Deshaies et al . , 1988; Sass et al . , 2003; Young et al . , 2003 ) , positioning them to play a role in recognizing misfolded mitochondrial proteins and targeting them to the UPS . On the other hand , we have found that the degradation of a native MOM protein , Fzo1 , largely does not require Hsp70 chaperones , extending to mitochondria a distinction between misfolded substrates and those whose ubiquitination occurs in a regulated manner . Whether there are other , yet to be identified , factors involved in the degradation of MOM proteins remains to be seen . Several mitochondrial inner membrane and IMS proteins have also been identified as proteasome substrates ( Bragoszewski et al . , 2013; Margineantu et al . , 2007; Pearce and Sherman , 1997; Radke et al . , 2008 ) . It now becomes of interest to ascertain how their degradation overlaps with and diverges from the QC pathway defined herein . Finally , it will also be important to determine how our findings extend to mammalian mitochondrial protein turnover and mitochondrial homeostasis as a whole .
Saccharomyces cerevisiae strains expressing ts- alleles were cultured at 25°C in minimal media supplemented with 2% glucose and the appropriate amino acids , unless otherwise indicated . For spot growth assays , 10-fold serial dilutions beginning with 0 . 1 OD600 units of cells were spotted to YPD and incubated at 25°C for 3 days or 37°C for 2 days . Strains used in this study can be found in the Supplementary file 1: Key Resources Table . Deletion collection strains were confirmed by PCR using a KanMX-specific oligo ( oMM19; see Supplementary file 1: Key Resources Table for oligo sequences ) paired with ORF-specific primers annealing 500 bp upstream of the start codon . A strain expressing genomic sam35-2HAts ( yMM37 ) was constructed by a one-step PCR-mediated HA tagging using pFA6a-3HA-His3M×6 ( Longtine et al . , 1998 ) as a PCR template with oligos oMM84 and oMM85 and integration into a sam35-2ts strain ( Li et al . , 2011 ) . Deletion strains yMM149 ( ubr1::KanMX ) and yJS155 ( ubx2::KanMX ) were constructed by PCR-mediated gene disruption using yeast deletion collection strains ( GE Dharmacon ) as PCR templates with oligos oMM236 and oMM237 , followed by integration into strain WCG4a ( for yMM149 ) or oligos oJS18 and oJS20 with integration into BY4741 ( for yJS155 ) . Strain yJS208 ( doa1::KanMX ) was constructed by PCR-mediated gene disruption using pFA6a-KanMX6 ( Longtine et al . , 1998 ) as a PCR template with oligos oMM257 and oMM258 and integration in strain WCG4a . Plasmids used in this study can be found in the Supplementary file 1: Key Resources Table . Plasmid pMM157 was constructed by PCR amplification of sam35-2HAts with adjacent promoter and terminator sequence from yMM37 using oligos oMM128 and oMM129 containing flanking XhoI and XbaI restriction sites , respectively , and ligation of this insert into the XhoI and XbaI sites in pRS315 ( Sikorski and Hieter , 1989 ) . Plasmid pMM160 was constructed by PCR amplification of sen2-1HAts with adjacent promoter and terminator sequence from yMM41 using oligos oMM129 and oMM130 containing flanking XbaI and XhoI restriction sites , respectively , and ligation of this insert into the XhoI and XbaI sites in pRS315 . Plasmids pMM231 and 234 were constructed by subcloning the Xho1/XbaI flanked insert from pMM157 and 160 , respectively , into pRS316 ( Sikorski and Hieter , 1989 ) . Plasmids pMD1 and pMD4 were constructed by digestion of pMM157 and pMM160 , respectively , with BamHI/AscI to replace the HA tag with GFP from pFA6a-GFP cut with the same sites . Plasmid pMM242 was generated in two steps . First , pUBX2-UBX2-TAP with flanking XhoI and AscI sites was PCR amplified using a Ubx2-TAP strain ( GE Dharmacon ) as a template with oligos oJS18 and oJS19 and ligated into pRS315 . The TAP tag was then dropped out by restriction digest with BamHI/AscI and replaced with annealed oligos oMM240 and oMM241 encoding a 3x FLAG epitope with overhang compatible for ligation into BamHI/AscI . pMM254 was generated by PCR amplifying the Doa1 promoter and ORF from the genome of BY4741 with oligos containing flanking XhoI ( oMM267 ) and AscI/3xFLAG epitope ( oMM268 ) and ligation into pRS426 ( Christianson et al . , 1992 ) digested at the same sites . Rabbit polyclonal anti-Sam35 ( Chan and Lithgow , 2008 ) was a generous gift from Trevor Lithgow . Rabbit polyclonal anti-Sis1 ( Yan and Craig , 1999 ) was a generous gift from Elizabeth Craig . Rabbit polyclonal anti-Cue1 and anti-ubiquitin were described previously ( Kostova et al . , 2009 ) . Commercial antibodies used were: mouse monoclonal PORIN ( MTCO1; abcam ) ; rabbit polyclonal Prc1 ( CPY; abcam ) ; mouse monoclonal GFP ( Santa Cruz Biotechnology ) ; mouse monoclonal phosphoglycerate kinase 1 ( PGK; Life Technologies ) ; rat monoclonal peroxidase-conjugated anti-HA ( 3F10; Roche ) ; mouse monoclonal anti-FLAG ( M2; Sigma-Aldrich ) ; rabbit polyclonal anti-FLAG ( Sigma-Aldrich ) ; and rabbit polyclonal anti-c-myc ( Abcam ) . Cycloheximide ( CHX ) chase analyses were performed as described previously Metzger et al . ( 2013 ) at 25°C or 37°C . For chases at 37°C , cells were cultured at the permissive temperature of 25°C until the addition of 100 μg/mL CHX , after which the temperature was increased to 37°C to accelerate the turnover of the ts- proteins . Maintaining cells at 25°C until the addition of CHX may be insufficient to fully inactivate ts- conditional yeast strains prior to the CHX chases , resulting in an underestimation of the role of the inactivated protein in degradation of substrates . Cells were then lysed in 1% β-mercaptoethanol ( βme ) /250 mM NaOH and proteins were precipitated in 5% trichloroacetic acid ( TCA ) . Protein pellets were resuspended in TCA sample buffer ( 3 . 5% SDS , 0 . 5 M DTT , 80 mM Tris pH8 . 8 , 8 mM EDTA , 15% glycerol , 0 . 1 mg/mL bromophenol blue ) . Samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies according to standard procedures . Proteins were detected using SuperSignal West Pico Luminol Enhancer Solution ( Thermo Scientific ) or Amersham ECL Select ( GE Healthcare ) and a G:box ( Syngene ) or c280 Imager ( Azure ) . Each CHX chase was repeated at least three times; shown in each figure is a representative blot . The percent of substrate remaining was calculated by quantification of anti-HA signal using ImageJ ( National Institutes of Health , Bethesda , MD ) , followed by normalization of this signal to the anti-PGK signal at the same time point . The ‘0’ time points were set to 100% and the mean and standard deviation ( SD ) at each time point for three independent biological replicates were graphed using GraphPad Prism 8 and fitted with one phase decay curves . Time points appearing to not have error bars have SD smaller than the size of the symbol . Mitochondria were isolated as described previously Gregg et al . ( 2009 ) , with the following changes for the analysis of ts- proteins . Cultures were grown in minimal media containing the appropriate amino acids at 25°C . Cells were incubated in DTT Buffer ( Gregg et al . , 2009 ) for 30 min at 25°C and in Zymolyase Buffer ( Gregg et al . , 2009 ) with Zymolyase-100T ( MP Biomedicals ) for 45 min at 25°C , after which time the resulting spheroplasts were washed and resuspended in an equal volume of Zymolyase Buffer without Zymolyase-100T , either at 25°C or prewarmed to 37°C , as indicated . CHX ( 100 μg/mL ) was added and spheroplasts were incubated without shaking at 25°C or 37°C for 5 min ( sen2-1HAts ) or 15 min ( sam35-2HAts ) . Spheroplasts were then homogenized using a glass homogenizer and the resulting cleared lysate was fractionated at 12 , 000xg into a post-mitochondrial supernatant fraction ( S ) and mitochondrial pellet ( P ) . Both fractions were precipitated in 10% TCA and washed in 2% TCA prior to resuspension in TCA sample buffer . Equivalent proportions of S and P were analyzed by SDS-PAGE and immunoblotting . For the isolation of purified mitochondria devoid of other organelles , mitochondrial pellets isolated as above were resuspended in 3 mL SEM buffer ( 10 mM Tris-HCl pH 7 . 4 , 0 . 6 M sorbitol , 1 mM EDTA , 0 . 2% BSA ) and overlaid on a sucrose gradient layered top to bottom with 6 mL 15% ( weight/volume ) sucrose/6 mL 23% sucrose/16 mL 32% sucrose/6 mL 60% sucrose and spun in a swinging-bucket rotor at 134 , 000xg for 1 hr at 4°C . The intact mitochondria residing at the 60%/32% sucrose interface were recovered and gently resuspended in SEM buffer and spun in a swinging-bucket rotor at 10 , 000xg for 30 min at 4°C . The pure mitochondrial pellet was precipitated in 10% TCA and washed in 2% TCA prior to resuspension in TCA sample buffer . For sodium carbonate ( Na2CO3 ) extraction of peripheral mitochondrial proteins , crude mitochondria isolated as above were treated with 0 . 2 M Na2CO3 or NaCl as described previously Boldogh and Pon ( 2007 ) and analyzed by immunoblotting . For ubiquitin visualization using anti-ubiquitin immunoblotting , 10–30 OD600 units of mid log phase ( OD600 = 0 . 8–1 ) cells were grown at 25°C and incubated at 37°C for 30 min prior to protein preparation using βme/NaOH/TCA , as described above . Protein pellets resuspended in TCA sample buffer were diluted in Dilution buffer ( 50 mM Tris-HCl pH 7 . 5 , 100 mM NaCl , 5 mM EDTA , 5% glycerol , 1% Triton X-100 , 1x Complete Protease Inhibitor Cocktail ( Roche ) , 1 mM NEM ) and HA-tagged substrates were isolated by immunoprecipitation ( IP ) for 18 hr at 4°C using mouse monoclonal anti-HA affinity matrix ( Sigma ) . Immunoprecipitated proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE . Ubiquitinated species were visualized by immunoblotting with ubiquitin antibodies . Unmodified substrates were detected by immunoblotting with HA antibodies . For ubiquitin visualization using anti-myc immunoblotting , 10–30 OD600 units of early log phase ( OD600 = ~0 . 4 ) cells were grown at 25°C , treated with 0 . 1 mM CuSO4 for 4 hr to induce expression of myc-ubiquitin , then incubated at 37°C for 30 min . Proteins were prepared as above . Mouse monoclonal anti-HA affinity matrix was pre-blocked with 1% ovalbumin and protein lysate was pre-cleared with Glutathione Sepharose 4-B ( GE Healthcare Life Sciences ) overnight at for 18 hr at 4°C , prior to IP of cleared protein lysate with the pre-blocked anti-HA affinity matrix for 4 hr at 4°C . Ubiquitinated species were visualized by immunoblotting with rabbit anti-c-myc antibodies . Unmodified substrates were detected by immunoblotting with HA antibodies . For TUBE isolation of ubiquitinated proteins , 30–100 OD600 units of mid log phase cell were grown at 25°C and incubated at 37°C for 30 min , at which time they were frozen at −80°C . Cell lysis was performed using glass beads in Lysis buffer ( 50 mM Tris-HCl pH 7 . 5 , 200 mM NaCl , 1% Triton X-100 , 1x Complete Protease Inhibitor Cocktail , 1 mM NEM ) . Lysates were cleared by centrifugation at 13 , 000xg for 20 min , then centrifugation at 13 , 000xg for 5 min . An aliquot of input lysate was precipitated in 10% TCA and protein pellets were resuspended in TCA sample buffer . The remainder of the lysate was incubated with TUBE-1 agarose ( Life Sensors ) for 18 hr at 4°C; ubiquitinated proteins were eluted from the TUBE-1 agarose with SDS-PAGE sample buffer . HA-tagged substrates in the input and TUBE-bound species were analyzed by SDS-PAGE and immunoblotting with HA antibodies . For analysis of ubiquitination from 12 , 000xg mitochondria and post-mitochondrial supernatant fractions , cells were grown at 25°C and treated with 0 . 1 mM CuSO4 for 2 hr to induce expression of myc-ubiquitin prior to crude mitochondrial fractionation as described in the previous section . S and P fractions were then subject to protein preparation and IP as described above for ubiquitin visualization using anti-myc immunoblotting . For co-IP , 10–30 OD600 units of mid log phase ( OD600 = 0 . 8–1 ) cells expressing Ubx2-FLAG were grown at 25°C and incubated at 37°C for 1 hr prior to glass bead lysis in Lysis Buffer ( 50 mM Tris-HCl pH7 . 5 , 100 mM NaCl , 5% glycerol , 1 mM DTT , 1 mM PMSF , 1x Complete Protease Inhibitor Cocktail , 1 mM NEM ) . Cleared lysate was added to anti-HA affinity matrix and immunoprecipitated for 18 hr at 4°C; bound proteins were eluted with SDS-PAGE sample buffer and analyzed by immunoblotting . For imaging Ubx2-GFP , logarithmically-growing yeast cells were immobilized on coverslips coated with concanavalin A and imaged using a Nikon Eclipse Ti inverted microscope , equipped with a 64 µm pixel CoolSNAP HQ2 camera ( Photometrics ) , Intensilight C-HGFIE illuminator , and 100x NA 1 . 42 Plan Apo objective . For live-cell analysis of ts- proteins , logarithmically growing cells were embedded in agarose and treated with 100 μg/mL CHX at 25°C ( time = 0 ) and then incubated at 37°C for the indicated times before imaging using a Nikon Eclipse Ti inverted microscope u60x NA 1 . 45 Plan Apo objective , Yokogawa spinning disc , 488 and 561 nm excitation lasers ( Agilent technology MCL-400 ) , back-illuminated EMCCD camera ( Andor , DU888 ) , and a 2x relay lens . A Nikon DS-U3 camera was used to record DIC images . 200 nm thick Z-sections spanning entire cell ( ~6 μm ) , were acquired . ImageJ ( National Institutes of Health ) was used to assemble the figures .
Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins . However , quality control ( QC ) pathways for misfolded mitochondrial proteins remain poorly defined . Here , we establish temperature-sensitive ( ts- ) peripheral mitochondrial outer membrane ( MOM ) proteins as novel model QC substrates in Saccharomyces cerevisiae . The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system . Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase ( E3 ) Ubr1 , while sam35-2HAts is ubiquitinated primarily by San1 . Mitochondria-associated degradation ( MAD ) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1 , providing the first evidence for chaperone involvement in MAD . In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex , Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor , Ubx2 , are implicated in their degradation . This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways .
Proteins are molecules that need to fold into the right shape to do their job . If proteins lose that shape , not only do they stop working but they risk clumping together and becoming toxic , potentially leading to disease . Fortunately , the cell has quality control systems that normally detect and remove misfolded proteins before they can cause damage to the cell . First , sets of proteins known as chaperones recognize the misfolded proteins , and then another class of proteins attaches a molecular tag , known as ubiquitin , to the misshapen proteins . When several ubiquitin tags are attached to a protein , forming chains of ubiquitin , it is transported to a large molecular machine within the cell called the proteasome . The proteasome unravels the protein and breaks it down into its constituent building blocks , which can then be used to create new proteins . Proteins are found throughout the different compartments of the cell and quality control processes have been well-studied in some parts of the cell but not others . Metzger et al . have now revealed how the process works on the surface of mitochondria , the compartment that provides the cell with most of its energy . To do this , they used baker’s yeast , a model laboratory organism that shares many fundamental properties with animal cells , but which is easier to manipulate genetically . The quality control process was studied using two mitochondrial proteins that had been mutated to make them sensitive to changes in temperature . This meant that , when the temperature increased from 25°C to 37°C , these proteins would begin to unravel and trigger the clean-up operation . This approach has been used previously to understand the quality control processes in other parts of the cell . By removing different quality control machinery in turn from the yeast cells , Metzger et al . could detect which were necessary for the process on mitochondria . This showed that there were many similarities with how this process happen in other parts of the cell but that the precise combination of chaperones and enzymes involved was distinct . Furthermore , when the proteasome was not working , the misfolded proteins remained on the mitochondria , showing that they are not transported to other parts of the cell to be broken down . In the future , understanding this process could help to find potential drug targets for mitochondrial diseases . The next steps will be to see how well these findings apply to human and other mammalian cells .