Herein, we show that Vms1 is part of an evolutionarily conserved mitochondrial stress-responsive system that promotes mitochondrial protein degradation and function. Vms1 is cytosolic in wild-type S. cerevisiae cells maintained in normal laboratory conditions. Vms1 translocates to mitochondria, however, in response to a variety of stress stimuli that all impact mitochondrial function. By analogy to the ERAD system that responds to unfolded proteins in the ER, accumulation of damaged, ubiquitinated or partially translocated proteins at mitochondria might signal for recruitment of the Vms1-Cdc48-Npl4 complex. Such a recruitment stimulus would be logical in light of the function of this complex in protein degradation as shown herein.
We provide several lines of evidence that the primary function of Vms1 is the mitochondrial recruitment of a complex containing Cdc48 and Npl4, thereby promoting ubiquitin-dependent protein degradation. First, mitochondrial perturbations cause mitochondrial translocation of Vms1 as demonstrated both by imaging of Vms1-GFP and by detection in gradient-purified mitochondria. Each of the experimental conditions under which the vms1Δ mutant exhibits impaired viability or growth, including rapamycin treatment, oxidative stress and static culture, cause mitochondrial translocation of Vms1. This suggests that the function of Vms1, with respect to these phenotypes, is performed at mitochondria.
Second, Vms1 is required for normal mitochondrial recruitment of Cdc48 and Npl4 in response to mitochondrial stress. Only a fraction of Cdc48 and Npl4 are recruited to mitochondria, presumably the same minor population that is Vms1-associated. If, as we propose, the phenotype of the vms1Δ mutant is due to a deficiency of Cdc48/ Npl4 in the vicinity of mitochondria, increasing the cellular concentration of these two proteins might rescue that growth defect. Indeed, overexpression of either Npl4 or Cdc48 partially rescued the vms1Δ growth defect.
Third, the primary function of Vms1 depends upon interaction with Cdc48. This observation was enabled by the discovery that Vms1 possesses an evolutionarily conserved VCP Interaction Motif (VIM). We showed that deletion of the yeast Vms1 VIM abrogated th e interaction with Cdc48. This same mutation completely destroyed the ability of Vms1 to confer rapamycin resistance despite having normal expression and mitochondrial localization. Mutation of three highly conserved residues in the human Vms1 VIM also destroyed its ability to interact with VCP. VIM-deleted Vms1 also failed to interact with Npl4, suggesting that the Vms1-Npl4 interaction is mediated by Cdc48.
Fourth, it appears that the population of Cdc48-Npl4 complex that is Vms1-associated is distinct from that which is associated with Ufd1. We have shown that both Vms1 and Ufd1 co-purify with Cdc48 and Npl4, and protein-protein interaction screens have shown that both interact with various components of the ubiquitin/ proteasome system, including Ufd2 and Ufd3 (Jensen et al., 2009
). In spite of this, Vms1 and Ufd1 do not co-purify in multiple experimental formats in our hands and there is no indication that Vms1 and Ufd1 interact in published datasets. Consistent with an important role in ERAD, protein interaction data suggest that Ufd1 interacts with Der1 (Jensen et al., 2009
), an ER membrane protein that is a central component of the ERAD machinery (Vembar and Brodsky, 2008
). In contrast, there is no indication of interaction of Vms1 with ER resident proteins and our data demonstrate mitochondrial localization of Vms1 under the conditions where the protein is most important for cell survival. The roles of Vms1 and Ufd1 in protein degradation are also distinct. The vms1
Δ mutant has impaired Fzo1 degradation, but under identical conditions, has modestly accelerated degradation of CPY*, which is almost absent in a ufd1-1
mutant. Genetically, both Cdc48 and Npl4 partially suppressed the vms1
Δ mutant phenotype, but Ufd1 overexpression had no effect. Based on these combined data, we suggest that the Vms1-associated Cdc48-Npl4 complex promotes mitochondrial protein degradation, while the Ufd1-associated proportion is required for the well-documented role of Cdc48 in ER protein degradation (Figure S7E
Fifth, Vms1 is required for normal ubiquitin-dependent protein degradation at mitochondria, specifically on the mitochondrial outer membrane. The best-characterized mitochondrial substrate of the ubiquitin/ proteasome system in yeast is the outer membrane protein Fzo1, and its degradation is impaired in the vms1Δ mutant. We suggest that Fzo1 is indicative of a broader impairment of ubiquitin-dependent protein degradation at the mitochondrial outer membrane and that impaired Fzo1 degradation is not a major cause of the vms1Δ mutant phenotypes we have observed. We, therefore, anticipate that the degradation of other proteins associated with the mitochondrial outer membrane will be found to be dependent on Vms1. Indeed, mitochondrial ubiquitin/ proteasome system-dependent protein degradation appears to be widely compromised in the vms1Δ mutant as evidenced by the altered accumulation of many poly-ubiquitinated proteins.
Sixth, loss of Vms1 increases cellular reliance on other, non-proteasomal modes of mitochondrial protein degradation. We observed enhanced mitophagy in the vms1
Δ mutant relative to wild-type, which was confirmed by examination of the steady state levels of a series of proteins from different mitochondrial compartments. The other major mode of mitochondrial protein degradation is enacted by intrinsic mitochondrial proteases, including Oma1 and Yme1. In the absence of Vms1, both Oma1 and Yme1 become almost completely essential for glycerol growth. These two pieces of data are strongly suggestive of a role for Vms1 in mitochondrial protein degradation. The coordination of proteasomal protein degradation and mitophagy and their role in maintaining mitochondrial function, preventing ROS accumulation and cell death has been also observed by others (Takeda et al., 2010
). The synthetic respiratory defect caused by loss of Vms1 and either Oma1 or Yme1, both of which reside in the mitochondrial inner membrane, raises an important question. Does Vms1, and by extension the ubiquitin-proteasome system, participate in the degradation of internal mitochondrial proteins? In mammalian cells, the intrinsic mitochondrial inner membrane proteins, UCP-2 and UCP-3, have both recently been shown to have unusually short half-lives, which are dependent upon the cytosolic proteasome (Azzu and Brand, 2010
; Azzu et al., 2010
). The OSCP subunit of the mitochondrial Complex V ATP synthase has also shown to be ubiquitinated and degraded by the cytosolic proteasome (Margineantu et al., 2007
). It is possible that a mitochondrial retrotranslocation system, analogous to that of the endoplasmic reticulum in ERAD, might extrude proteins for degradation in the cytosol.
Finally, the aberrant cellular physiology of the vms1
Δ mutant suggests that the Vms1 protein is required for maintenance of mitochondrial function. The loss of Vms1 causes a marked time-dependent failure of mitochondrial respiration. Concurrently, we also observed an increase in oxidative stress and its damaging effects. Likely as a direct consequence, the vms1
Δ mutant exhibits progressively more pronounced cell death in static culture. Interestingly, these phenotypes are strikingly similar to that observed for the S565G mutant of Cdc48 (Braun et al., 2006
; Madeo et al., 1997
), which fails to stably interact with Vms1. Therefore, two independent genetic manipulations, mutation of Cdc48 and deletion of Vms1, that prevent the Vms1-dependent regulation of Cdc48 both cause cell death with similar mitochondrial sequellae. The importance of mitochondrial protein quality control for mitochondrial function and healthy lifespan has been recently emphasized by studies of the mitochondrial matrix Lon protease (Luce and Osiewacz, 2009
Based on these genetic and biochemical connections, we propose a model wherein mitochondrial stress causes the recruitment of a subpopulation of Cdc48 and Npl4 to mitochondria through their interaction with Vms1 (Figure S7E
). We suggest that the Vms1-dependent translocation to mitochondria enables Cdc48 and its cofactor Npl4 to perform a function on mitochondria that is similar to its function in ERAD. In the absence of Vms1, damaged, misfolded and ubiquitinated proteins accumulate causing progressive mitochondrial dysfunction and eventually cell death.
These data and the high degree of conservation throughout eukaryotes suggest that Vms1 performs similar functions in higher eukaryotes. We propose that Vms1 is a component of an evolutionarily conserved system for maintaining mitochondrial function through protein quality control. In its absence, progressive mitochondrial dysfunction causes shortened lifespan as observed in yeast and worms. Due to the central role for mitochondrial dysfunction in age-related human diseases, including neurodegenerative diseases, we consider it likely that alterations in Vms1 expression, activity or associations would impact the incidence of such pathologies. Indeed, mutations in VCP, the human ortholog of Cdc48, cause progressive muscle weakness and frontotemporal dementia (Watts et al., 2004
; Weihl et al., 2009
). Of more direct interest, a locus conferring susceptibility to Alzheimers disease has been mapped to human chromosome 2q (Holmans et al., 2005
), with a second study mapping susceptibility to the immediate vicinity of the human VMS1
ortholog (Scott et al., 2003
). It will be important to define whether these susceptibility loci are related to alterations in Vms1 function. A more detailed understanding of the Vms1 system could aid in understanding the mitochondrial etiology of disease and the cellular systems to prevent it.