A Stress-Responsive System
for Mitochondrial Protein Degradation
Jin-Mi Heo,1Nurit Livnat-Levanon,2,8Eric B. Taylor,1,8Kevin T. Jones,3,8,9Noah Dephoure,4Julia Ring,5Jianxin Xie,6
Jeffrey L. Brodsky,7Frank Madeo,5Steven P. Gygi,4Kaveh Ashrafi,3Michael H. Glickman,2and Jared Rutter1,*
1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
2Department of Biology and The Russell Berrie Nanotechnology Institute, Technion, Israel Institute of Technology, 32000 Haifa, Israel
3Department of Physiology and UCSF Diabetes Center, University of California, San Francisco, San Francisco, CA 94158-2517, USA
4Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA
5Department of Microbiology, Institute of Molecular Biosciences, Karl-Franzens-University of Graz, Graz, Austria
6Cell Signaling Technology, Inc., Danvers, MA 01923, USA
7Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
8These authors contributed equally to this work
9Present address: Department of Molecular, Cell, and Developmental Biology, University of California,
Los Angeles, Los Angeles, CA 90095, USA
We show that Ydr049 (renamed VCP/Cdc48-associ-
ated mitochondrial stress-responsive—Vms1), a
member of an unstudied pan-eukaryotic protein
family, translocates from the cytosol to mitochondria
upon mitochondrial stress. Cells lacking Vms1 show
progressive mitochondrial failure, hypersensitivity
to oxidative stress, and decreased chronological life
span. Both yeast and mammalian Vms1 stably
interact with Cdc48/VCP/p97, a component of the
ubiquitin/proteasome system with a well-defined
role in endoplasmic reticulum-associated protein
degradation (ERAD), wherein misfolded ER proteins
are degraded in the cytosol. We show that oxidative
stress triggers mitochondrial localization of Cdc48
and this is dependent on Vms1. When this system is
impaired by mutation of Vms1, ubiquitin-dependent
mitochondrial protein degradation, mitochondrial
respiratory function, and cell viability are compro-
mised. We demonstrate that Vms1 is a required
component of an evolutionarily conserved system
for mitochondrial protein degradation, which is
necessary to maintain mitochondrial, cellular, and
Mitochondria are dynamic and complex organelles that are
essential for many aspects of cellular function including metab-
olism and cell death. Consistent with these critical roles, mito-
chondrial dysfunction is associated with most aging-related
human diseases, including neurodegenerative disorders, type
2 diabetes, and cancer (Wallace, 2005). The best current inven-
tory of mammalian mitochondrial resident proteins consists of
1098 proteins (Pagliarini et al., 2008). Surprisingly, nearly 300
of these proteins have completely undefined functions, including
many that are highly conserved throughout eukarya, indicating
that they perform a fundamental and important function
(Meisinger et al., 2008; Pagliarini et al., 2008). The genes that
encode the mitochondrial proteome are heavily represented
among known human disease genes, with about 20% of pre-
dicted human mitochondrial proteins implicated in one or more
hereditary diseases (Andreoli et al., 2004; Elstner et al., 2008).
Presumably, the quarter of the mitochondrial proteome that is
uncharacterized contains other proteins with links to human
disease that await discovery. Making these connections would
be greatly facilitated by an understanding of the biochemical
and physiological function of these proteins.
Therefore, we initiated studies to determine the genetic and
biochemical functions of a subset of these conserved but
uncharacterized mitochondrial proteins (Hao and Rutter, 2009).
As a result of this project, we previously identified the unstudied
Yol071 yeast protein, which we named Sdh5, as a critical
assembly factor for the succinate dehydrogenase complex/
complex II (Hao et al., 2009). By virtue of this observation, we
identified the human SDH5 ortholog as the causative gene in
a familial form of the paraganglioma neuroendocrine tumor
syndrome (Hao et al., 2009). We describe herein another
unstudied conserved mitochondrial protein, Ydr049, which we
now designate VCP/Cdc48-associated mitochondrial stress-
responsive 1 (Vms1). VMS1 is highly evolutionarily conserved,
with one ortholog existing in most eukaryotic species. Initially
using yeast, we show that Vms1 protects mitochondrial respira-
tory function and combats cell death in response to various
stress stimuli. Both yeast and human Vms1 copurify with
Cdc48/VCP/p97, and we show that Vms1 stably associates
with both Cdc48 and its cofactor Npl4, which have well-defined
roles in the degradation of endoplasmic reticulum (ER) proteins
dria is Vms1 dependent and that this system is required for
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 465
Clear links between mitochondria, which are membrane-
confined organelles, and the cytosolic ubiquitin/proteasome
system have recently been described (Livnat-Levanon and
Glickman, 2010). Fzo1,a mitochondrial outer membraneprotein,
was shown to be ubiquitinated by the Mdm30 cytosolic E3 ubiq-
uitin ligase and degraded by the proteasome (Cohen et al., 2008;
Fritz et al., 2003). Mitochondria in cells lacking MDM30 aggre-
gate in clumps and respire inadequately, leading to shortened
life span in response to stress. This is likely a manifestation of
a broader system for degradation of mitochondria-associated
proteins. The flux of imported proteins and the proximity to
oxidative phosphorylation result in significant protein damage
and misfolding at mitochondria, necessitating a responsive
quality control system. The mitochondria contains an intrinsic
system of proteases dedicated to quality control (Tatsuta,
2009), but the cytosolic ubiquitin/proteasome system appears
to also play a role. Based on the data presented herein, we
propose that Vms1 plays a conserved role in recruiting the
ubiquitin/proteasome system for stress-responsive mitochon-
drial protein degradation.
Vms1 Necessity and Mitochondrial Translocation under
Conditions of Mitochondrial Stress
The Vms1 protein was detected by mass spectrometry in highly
purified mitochondria (Sickmann et al., 2003). Surprisingly,
however, a functional Vms1-GFP fusion localized primarily to
the cytosol in synthetic glucose-containing medium (Figure 1A,
top) as well as rich and synthetic media containing glycerol or
raffinose (data not shown). In a small fraction of cells grown in
synthetic glucose medium, Vms1-GFP partially colocalized
with the mitochondrial marker mito-RFP (Figure 1A, top, marked
with arrow). Hypothesizing that this small population might be
cells that have lost mitochondrial DNA (rho?), we directly tested
a rho?strain and found that Vms1 was partially mitochondrial
in all cells (Figure 1A). Loss of mitochondrial DNA has a number
of effects on mitochondrial physiology, including decreased
mitochondrial membrane potential (Petit et al., 1996). The
combination of Antimycin A and oligomycin, which blocks the
establishment of the mitochondrial membrane potential (Priault
et al., 2005), caused near-complete localization of Vms1 to
mitochondria (see Figure S1A available online). Treatment with
the uncouplers CCCP or FCCP, which directly dissipate the
membrane potential, also caused mitochondrial localization of
Vms1 (Figure S1A).
We also found that oxidative stress, which indirectly impairs
mitochondrialfunction, elicited byhydrogen peroxide or deletion
of the mitochondrial superoxide dismutase, Sod2, caused
mitochondrial translocation of Vms1 (Figure 1A and data not
shown). Hydrogen peroxide also caused Vms1-GFP localization
to punctae that do not have mito-RFP, which may be damaged
mitochondria that fail to import mito-RFP. Treatment with the
TOR protein kinase inhibitor rapamycin, which increases mito-
chondrial oxidative damage (Kissova et al., 2006), also caused
robust mitochondrial translocation of Vms1-GFP (Figure 1A).
We hypothesize that perturbation of mitochondrial function is
the proximal signal that causes Vms1 translocation, with rapa-
mycin and oxidative stress acting indirectly through mitochon-
drial oxidative damage.
To address the functional significance of Vms1 mitochondrial
localization, we analyzed the growth of the vms1D mutant under
conditions that cause Vms1 mitochondrial translocation. While
the vms1D mutant was indistinguishable from wild-type in
normal conditions, it exhibited severe hypersensitivity to
hydrogen peroxide, an effect exacerbated when combined
with loss of the mitochondrial Sod2 (Figure 1B). In fact, the
sod2D vms1D double mutant exhibited a modest growth defect
even on normal medium (Figure 1B, top). Similarly, the vms1D
mutant failed to grow in the presence of rapamycin, which was
completely rescued by plasmid-borne VMS1 and partially
rescued by expression of the human VMS1 gene (Figure 1C).
This failure to grow is a result of cell death caused by rapamycin
treatment (Figure S1B). A high-copy suppressor screen yielded
three genes that, when provided in high copy, rescue the
vms1D mutant rapamycin hypersensitivity: XBP1, GRX3, and
ZWF1 (Figures S1C and S1D). Each of the three has a role in
response to stress, particularly oxidative stress. Xbp1 is
a stress-induced transcription factor. Grx3 is a glutaredoxin
and is a component of a major antioxidant system. Loss of
Grx3 leads to increased protein oxidation and hypersensitivity
to oxidizing agents (Rodriguez-Manzaneque et al., 1999).
duces NADPH, which is essential for the reduction of oxidized
glutathione. The zwf1D mutant is hypersensitive to oxidative
stress agents, particularly hydrogen peroxide (Nogae and John-
ston, 1990; Outten and Culotta, 2003). These data suggest that
Vms1 is required for normal tolerance to oxidative stress and
this is manifest by hypersensitivity to both hydrogen peroxide
Deletion of VMS1 Causes Loss of Mitochondrial
Function and Viability
During the course of these experiments, we observed that
cultures maintained past log phase exhibited mitochondrial
Vms1 in the absence of other stressors. For example, mainte-
drial localization of Vms1-GFP (Figure 2A). We confirmed the
mitochondrial localization of Vms1-HA by biochemical fraction-
ation after mild crosslinking. Vms1 was detectable in both the
cytosolic and crude mitochondrial fractions, and a portion of
Vms1-HA comigrated exactly with the mitochondrial marker
Tom20 in a sucrose gradient (Figure 2B).
As the vms1D mutant grows poorly under the same conditions
that cause mitochondrial translocation of Vms1, we hypothe-
failure. We tested mitochondrial respiration, and the wild-type
and vms1D strains consumed oxygen equivalently in log phase.
After 1.5 days of culture, however, the vms1D mutant had
a significant impairment in oxygen consumption (Figure 2C).
Due to an exposed iron-sulfur cluster, the activity of aconitase
is exquisitely sensitive to mitochondrial oxidative stress. As in
the respiration assay, there was no difference in aconitase
activity in log phase, but the activity of the vms1D mutant was
significantly reduced relative to wild-type at day 1.5 without
a reduction in protein level (Figure 2D and data not shown).
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466 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
We hypothesize that the progressive mitochondrial stress in
stationary phase culture necessitates Vms1 for the maintenance
of respiration and aconitase activity.
For cells in stationary phase, mitochondrial respiration is
required for ATP synthesis and cell survival (Werner-Washburne
et al., 1993). Therefore, we sought to determine whether the
time-dependent loss of mitochondrial function in the vms1D
mutant might be manifest as a time-dependent defect in colony
formation on glycerol medium, wherein ATP generation and
growth requires mitochondrial respiration. At day 1.5 of culture,
glycerol growth of the vms1D strain remained similar to the
wild-type level (Figure 2E). This suggests that the mitochondrial
impairment observed at day 1.5, as manifest by decreased
oxygen consumption and aconitase activity, is reversible at
this point. In contrast, the vms1D mutant was greatly impaired
in glycerol colony formation at day 3.5 (Figure 2E). By day 5.5,
+ H O
WT + ev
SD-Ura + rapamycin
WT + ev
SD - Ura
Figure 1. Vms1 Exhibits Stress-Responsive Mitochondrial Translocation, and Loss of Vms1 Causes Hypersensitivity to Hydrogen Peroxide
(A) The vms1D strain containing both a plasmid expressing mito-RFP (a fusion of the N. crassa Su9 presequence to RFP) and a plasmid expressing Vms1-GFP
under the native VMS1 promoter was grown in SD-Ura-Leu medium. Upon reaching mid-log phase, the culture was either treated with vehicle (top) or with
compounds as indicated and subjected to fluorescence microscopy. The second row shows representative images of Vms1-GFP localization in an vms1D
rho?strain (lacking the mitochondrial genome) in log phase. The field shown in the top image was selected to show the weak mitochondrial localization of
Vms1-GFP in the absence of stressor in a small percentage of cells (indicated with an arrow). Representative images are shown.
SD-Ura +3 mM hydrogen peroxide (bottom) plates and grown at 30?C for 2 days.
gene under the control of the yeast GPD promoter (phVMS1). Each strain was streaked on an SD-Ura plate without (top) or with 30 ng/ml rapamycin (bottom) and
grown at 30?C for 2 days (top) or 5 days (bottom).
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Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 467
Log Day 1.5
Aconitase activity (OD/sec/mg)
WT + e.v.
vms1Δ + e.v.
sod2Δ + e.v.
vms1Δ sod2Δ + e.v.
SGly - Ura
Day 1 Day 3Day 8
Colony Forming Units/500 cells
1.5 days of culture
Cyt mito 1 2 3 4 5 6 7 8 9 10 Fr #
Figure 2. Deletion of VMS1 Induces Mitochondrial Dysfunction
(A) The vms1D strain containing plasmids expressing mito-RFP and Vms1-GFP was grown in SD-Leu-Ura for 1.5 days and subjected to fluorescence imaging.
(B) The vms1D strain containing a plasmid expressing C-terminally HA-tagged Vms1 was grown for 1.5 days and subjected to differential centrifugation. The
mitochondria-enriched fraction (mito) was then fractionated on a sucrose cushion and subjected to SDS-PAGE followed by western blot. Tom20 and Pgk1
were used as mitochondrial and cytoplasmic markers, respectively.
(C) and aconitase activity assay (D). Mean ± SD of three independent cultures is shown.
(E) WT and vms1D strains in the W303 background, grown in synthetic complete glucose (SD) media for 1.5, 3.5, 5.5, or 8.5 days were 5-fold serially diluted, and
equivalent cell numbers of each strain were spotted on both YPAD and YPAGlycerol plates and grown at 30?C.
(F) WTand twoindependentvms1DstrainsintheBY4741 background weregrown insyntheticcompleteglucose(SD) mediafor 1,3,or8days.Five hundredcells
from each culture were plated on YPAD and grown at 30?C for determination of colony formation. Colony-forming units were determined for at least three inde-
pendent cultures per strain, and mean ± SD is shown.
Mitochondrial Stress and Protein Degradation
468 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
colony formation on glycerol was nearly absent (Figure 2E). After
8.5 days of culture, the vms1D mutant also showed greatly
reduced ability to form colonies on glucose medium (Figure 2E).
This is likely due to cell death, as we obtained similar results
using exclusion of trypan blue as a measure of viability (data
not shown). Two independent vms1D isolates in a different strain
background (BY4741) exhibited an even more rapid loss of
viability than was observed in the W303 background (Figure 2F).
Oxidative stress has been proposed to be the key mediator in
causing cell death in static culture (Fabrizio et al., 2001), and
the vms1D mutant showed a significant increase in the oxidation
of DHE to ethidium (Figure 2G and Figure S2A).
In addition to measuring mitochondrial function and glycerol
growth in static culture, we also examined the effect of oxida-
tive stress. As with growth to day 1.5, combination with the
sod2D mutant caused a significant loss of oxygen consumption
and aconitase activity in the vms1D mutant (Figures S2B and
S2C). Similarly, while the vms1D mutant exhibited normal glyc-
erol growth in the absence of additional stressors, combination
with a deletion of SOD2 caused a complete loss of growth on
glycerol medium (Figure 2H). Combined, these data show that
the vms1D mutant has severely compromised mitochondrial
activity under the conditions that cause Vms1 mitochondrial
VMS1 Localization and Function in C. elegans
To determine whether VMS1 orthologs from other eukaryotes
function similarly, we examined vms-1 in C. elegans, which
contains a single VMS1 ortholog encoded by K06H7.3. Either
of two nonoverlapping RNAi constructs targeting vms-1 caused
a marked reduction in viability in response to hydrogen peroxide
(Figure 3A). The surviving vms-1-depleted individuals were
dramatically more lethargic than controls. This was confirmed
in a mutant line carrying a deletion predicted to remove the
majority of the VMS-1 protein (data not shown). Under standard
growth conditions, vms-1 mutants and RNAi-treated animals
had normal morphology and wild-type growth and development.
Activation of numerous stress-response genes, including the
mitochondrial superoxide dismutase genes sod-2 and sod-3, is
dependent on the function of the insulin-regulated FOXO tran-
scription factor DAF-16 (Murphy et al., 2003; Oh et al., 2006).
As expected, daf-16 mutant animals were also hypersensitive
to hydrogen peroxide treatment (Figure 3A). Treatment of daf-
16 mutants with vms-1 RNAi caused further hypersensitivity of
similar relative magnitude to that observed in wild-type vms-1
RNAi-treated animals (Figure 3A). Knockdown of vms-1 also
caused a significant decrease in life span of wild-type animals
and a further decrease in the already shortened life span of
daf-16 mutants (Figure 3B). These findings suggest that vms-1
functions in parallel with insulin signaling to regulate stress resis-
tance and life span.
To determine the tissue and subcellular expression patterns of
C. elegans VMS-1, we generated transgenic lines in which full-
length VMS-1 fused to a GFP reporter was expressed from the
native vms-1 promoter. This reporter was expressed broadly
during embryonic development. In larval stages and in adults,
expression was noted in intestinal cells, specific neurons in the
head and the tail, and in the ventral nerve cord (Figure 3C and
Figure S3). In untreated animals, VMS-1::GFP localized to the
cytoplasm in intestinal cells and neurons. In head amphid
neurons, VMS-1::GFP was uniformly detected in the dendritic
processes, where it was specifically excluded from mitochon-
dria, as determined by lack of colocalization with DIC-
1::mCherry (Kass et al., 2001) (Figure 3C). Exposure of animals
to hydrogen peroxide, however, caused colocalization of VMS-
1::GFP with DIC-1::mCherry (Figure 3C), an identical pattern to
that seen for other mitochondrial proteins (Hu and Barr, 2005).
Together, these findings indicate that, as in yeast, C. elegans
vms-1 function is dispensable for viability and growth but is
required for protection against oxidative stress and for wild-
type life span.
Vms1 Constitutively Interacts with Cdc48 and Npl4
While the Vms1 protein expressed in E. coli migrated as a mono-
mer in gel filtration chromatography, endogenous Vms1 from
crude yeast lysates migrated in a large >500 kDa complex
(data not shown). To identify subunits of the putative Vms1
complex, we purified a functional Vms1-TAP fusion and identi-
fied associated proteins by mass spectrometry. Cdc48,
a hexameric AAA-ATPase with a well-studied role in protein
degradation (Jentsch and Rumpf, 2007), copurified almost stoi-
chiometrically with Vms1-TAP (Figure 4A).
The Vms1-Cdc48 interaction was confirmed by coimmuno-
precipitation of epitope-tagged versions of Vms1 and Cdc48 ex-
pressed under their endogenous promoters. Immunoprecipita-
tion of Vms1-HA pulled down Cdc48-myc (Figure 4B). This
interaction was alsoobservedfollowing 2hrtreatment with rapa-
mycin (Figure S4A), a condition that causes Vms1 mitochondrial
translocation. The S565G mutant of Cdc48 was previously
reported to cause increased sensitivity to oxidative stress,
reduced respiratory activity, and increased cell death (Braun
et al., 2006; Madeo et al., 1997). This mutant was expressed at
wild-type levels, but interacted much more weakly with Vms1
than did wild-type Cdc48 (Figure 4B). Thus, Vms1 exists in
a stable complex with Cdc48, and this interaction is disrupted
by a Cdc48 mutation associated with increased oxidative stress
sensitivity and cell death. Cell death in Cdc48-S565G mutant
strains has been previously shown by increased annexin V and
propidium iodide (PI) staining (Madeo et al., 1997). Like Cdc48-
S565G mutant strains, the vms1D mutant strain had a signifi-
cantly increased fraction of Annexin V and PI-positive cells
(Figures S4B and S4C).
fluorescence was determined by FACS analysis for each strain. For each strain, three independent cultures were tested and mean ± SD is shown.
(H) WT, vms1D, sod2D,and vms1D sod2Dstrains were transformedwithempty vector (ev), aplasmid containing the SOD2 gene (pSOD2) or aplasmid containing
theVMS1gene (pVMS1).Eachstrain wasgrown tosaturationinSD-Uramedia.Serial5-fold dilutions ofeachculturewerethenspotted onanSGlycerol-Uraplate
and grown at 30?C.
Mitochondrial Stress and Protein Degradation
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 469
Cdc48 is a component of the ubiquitin/proteasome system
and promotes protein degradation in a variety of cellular
contexts, including membrane fusion, cell-cycle regulation, tran-
scription factor activation, and ERAD (Bays and Hampton, 2002;
Ye, 2006). To carry out these diverse functions, Cdc48 interacts
with different cofactor proteins that target Cdc48 activity to
distinct cellular sites and also mediate ubiquitin and proteasome
binding (Jentsch and Rumpf, 2007). We found that Vms1 also
associated with the Cdc48 cofactor Npl4 (Figure 4C) and this
interaction was maintained upon rapamycin treatment (Fig-
ure S4A). In the same experiment, however, we could not detect
an interaction between Vms1 and Ufd1, another Cdc48 cofactor
that has been implicated, along with Npl4, in the activity of
Cdc48 in ERAD (Figure 4C). To confirm the lack of interaction
with Vms1, we tested the ability of Ufd1 to interact with Vms1
a strong interaction with Npl4 as expected, it failed to interact
0510 1520 25
Days of adulthood
N2 RNAi 1
N2 RNAi 2
daf-16 RNAi 1
daf-16 RNAi 2
Figure 3. C. elegans vms-1 Is Required for
Wild-TypeLife Span andHydrogenPeroxide
(A) Wild-type (N2) or daf-16 mutant worms were
grown to the first day of egg laying on bacteria
expressing vector control or either of two indepen-
scored after 5 hr in 20 mM H2O2at room tempera-
ture. Results shown are the average ± SD of three
replicate experiments comprised of 100 animals
each. *p < 0.05 and **p < 0.01.
(B) Wild-type (N2) or daf-16 mutant worms grown
were as follows: wild-type on vector control was
18.0 ± 0.1 days, wild-type on RNAi 1 was
15.5 ± 0.2 days (p < 0.0001), wild-type on RNAi 2
was 15.0 ± 0.2 days (p < 0.0001), daf-16 on vector
control was 12.1 ± 0.1 days, daf-16 on RNAi 1 was
10.3 ± 0.1 days (p < 0.0001), and daf-16 on RNAi 2
was 9.3 ± 0.1 days (p < 0.0001). p values reflect
statistical significance of each RNAi treatment
compared to vector control for each strain.
(C) Subcellular localization of VMS-1::GFP in
dendritic processes of amphid neurons was
imaged in worms coexpressing Pvms-1::VMS-
marker (Pegl-3::DIC-1::mCherry). Worms were
treated for 1 hr in either M9 as a control (top) or
tative images for each population are shown.
Three sites of mito-mCherry localization and
VMS-1::GFP exclusion in the control images are
indicated with arrows.
with Vms1 (Figure 4D). We therefore
speculate that Vms1 and Ufd1 are mutu-
ally exclusive components of the Cdc48-
Npl4 complex. This idea is corroborated
by high-throughput protein-protein inter-
action analyses that show both Vms1
and Ufd1 as interacting with Cdc48 and
Npl4, but no interaction between Vms1
and Ufd1 (Jensen et al., 2009).
The Vms1 C-terminal region (Figure S4D) was necessary and
sufficient for Cdc48 interaction (Figure 4E). For an unknown
reason, deletion of the N terminus actually resulted in increased
interaction with Cdc48 relative to wild-type Vms1. The Vms1
interaction with Npl4 exhibited the exact same pattern of domain
dependence (Figure 4F). We identified a sequence at the
extreme C terminus of Vms1 that showed similarity with the
VCP (mammalian Cdc48 ortholog) interaction motif, or VIM,
found in the human E3 ubiquitin ligase GP78 and SVIP proteins
(Yeung et al., 2008). The putative VIM sequence in yeast Vms1 is
highly conserved in Vms1 orthologs (Figure 5A). A mutant lack-
ing this motif (as indicated in Figure 5A) exhibited a wild-type
pattern of localization—cytosolic in normal conditions, with
peroxide (Figure S5). This mutant, however, completely failed
to interact with either Cdc48 or Npl4 (Figures 5B and 5C),
suggesting that Vms1 interacts directly with Cdc48 through
Mitochondrial Stress and Protein Degradation
470 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
a C-terminal VIM sequence and the interaction with Npl4 is
mediated by Cdc48.
us to test whether the genetic function of Vms1 requires interac-
tion with Cdc48 and Npl4. We assayed the ability of wild-type
and VIM mutant VMS1, expressed from the native promoter, to
rescue the rapamycin hypersensitivity of the vms1D mutant
strain. As shown in Figure 5D, wild-type Vms1 rescued fully,
but the VIM mutant Vms1 had no effect on growth despite being
These data suggest that the function of Vms1 required for rapa-
mycin resistance, which we hypothesize relates to protection of
mitochondrial function from oxidative damage (see Figure 2), is
completely dependent upon interaction with Cdc48.
To identify the protein interactions of mammalian Vms1, we
conducted a tandem-affinity purification of a Flag/HA-tagged
mouse Vms1 expressed in mouse C2C12 cells. The final elution
had essentially only two bands (Figure 5E), Vms1-Flag/HA and
VCP, the mammalian ortholog of Cdc48. This interaction was
confirmed by coimmunoprecipitation experiments. While wild-
type Vms1 precipitated endogenous VCP (Figure 5F, lane 6),
this interaction was completely lost in a mutant wherein three
highly conserved VIM residues (as indicated in Figure 5A) had
been mutated to alanine (Figure 5F, lane 9). This experiment
was performed quantitatively to enable determination of the
amount of VCP that associates with Vms1. While greater than
90% of the tagged Vms1 was precipitated, we observed essen-
tially no depletion of the total VCP (Figure 5F, compare lanes
4–6), suggesting that the Vms1-associated VCP accounts for
less than 5% of the total cellular VCP. The yeast Vms1 also
appears to interact with a similarly small fraction of the total
cellular Cdc48 (data not shown). This is consistent with the
observation that Cdc48 has a much higher abundance than
Vms1 in normal yeast cells (Ghaemmaghami et al., 2003).
Vms1-Dependent Mitochondrial Translocation of Cdc48
Based on the stable and constitutive interaction between Vms1
and both Cdc48 and Npl4, we hypothesized that Vms1 might
mediate the recruitment of a subset of Cdc48 and Npl4 to mito-
chondria. Under normal conditions, Cdc48-GFP localized
throughout the cytoplasm, nucleus, and ER (Figure 6A). Upon
hydrogen peroxide treatment, however, a fraction of Cdc48-
GFP translocated to mitochondria in wild-type cells (Figure 6A).
The stress-induced mitochondrial translocation of Cdc48 was
nearly absent in a vms1D mutant strain, but was restored by
a plasmid-borne copy of VMS1 (Figure 6A). Cdc48-GFP also
exhibited localizationto nonmitochondrial
hydrogen peroxide treatment that was independent of Vms1.
The ratio of Cdc48-GFP that colocalized with mito-RFP versus
total cellular Cdc48-GFP was blindly quantitated in these three
strains in the presence and absence of hydrogen peroxide.
GFP (Figure 6B). There appears to be, however, a small fraction
of Cdc48 that shows stress-responsive mitochondrial colocali-
zation that is independent of Vms1. Our imaging and quantifica-
tion are currently unable to distinguish whether this is due to an
alternative mitochondrial-targeting system or to Cdc48 localiza-
tion to sites nearby mitochondria. A fraction of Npl4 also exhibits
stress-responsive translocation to mitochondria that is depen-
dent on Vms1 (Figure S6).
If Vms1 recruits Cdc48 and Npl4 to increase their local
concentration at mitochondria in response to stress, overex-
pression of Npl4 and/or Cdc48 might partially complement the
vms1D mutant phenotype by nonspecifically increasing their
local concentration at mitochondria. As expected, both Cdc48
tivity of the vms1D mutant strain (Figure 6C). Consistent with the
observation that Ufd1 does not associate with Vms1, overex-
pression of Ufd1 had no effect on the vms1D mutant phenotype
(Figure 6C). Together, these data support a role for Vms1 in
recruiting Cdc48 and Npl4 to mitochondria in response to stress,
and this activity is a primary function of Vms1.
rotranslocation of lumenal ER or ER membrane proteins to the
cytosol where they are degraded by the proteasome in a ubiqui-
tin-dependent manner (Raasi and Wolf, 2007; Vembar and Brod-
sky, 2008). Under the hypothesis that the Vms1-Cdc48-Npl4
complex functions similarly at mitochondria, we measured the
steady-state levels of Fzo1. Fzo1 is a mitochondrial outer
membrane protein that is the best-characterized mitochondrial
target of ubiquitin/proteasome system-dependent degradation
in yeast (Cohen et al., 2008; Escobar-Henriques et al., 2006).
While the levels of Fzo1 are identical in wild-type and the
vms1D mutant in log phase (data not shown), the vms1D mutant
exhibited elevated Fzo1 levels at day 2.5 of culture (Figure 7A).
The levels of Fzo1 were similar to that found in a mutant lacking
Mdm30, the principal ubiquitin E3 ligase involved in Fzo1 degra-
dation (Cohen et al., 2008). The mdm30D vms1D double mutant
showed an additive effect (Figure 7A).
If the Vms1-Cdc48-Npl4 complex is required for normal Fzo1
degradation, mutants of Cdc48 and Npl4 should also show
elevated Fzo1 steady-state levels. The S565G mutant of
Cdc48, which exhibits impaired Vms1 binding, had elevated
Fzo1 levels similar to the vms1D mutant (Figure 7B). The Fzo1
levels of the vms1D cdc48-S565G double mutant are similar to
the two single mutants, consistent with the proteins acting within
the same pathway to promote Fzo1 degradation. Fzo1 also
accumulated in the temperature-sensitive npl4-1 mutant at the
permissive temperature (Figure S7A), where there is no growth
defect on glucose-containing medium (see Figure S7B). Consis-
tent with a role for Npl4 in supporting mitochondrial function, the
npl4-1 mutant grew poorly on glycerol medium, but not glucose
medium (Figure S7B).
To directly address the stability of Fzo1, we examined the
degradation kinetics of an Fzo1-HA fusion protein following
cycloheximide addition to prevent new protein synthesis. Degra-
dation of Fzo1-HA was reproducibly delayed in the vms1D
mutant relative to wild-type cells (Figure 7C—quantitation in
Figure S7C). Under identical experimental conditions, the
ERAD substrate CPY* was degraded with similar kinetics in
the wild-type and vms1D mutant strains (Figure 7D). In fact, the
degradation of CPY* in the vms1D mutant was slightly enhanced
relative to wild-type (Figure 7D), while CPY* was stabilized in
Mitochondrial Stress and Protein Degradation
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 471
Vms1-HA − + − +
Ufd1-HA − + − +
Figure 4. Vms1 Constitutively Interacts with Cdc48 and Npl4
(A) Strainsexpressingeither untagged or TAP-tagged Vms1underthenative VMS1 promoterweregrown to late logphase and subjected toTAP purification. The
final eluates from each of two independent purifications for each strain were analyzed by SDS-PAGE and Coomassie staining. The major unique bands from the
TAP-tagged Vms1 purification were identified by mass spectrometry as Cdc48 and Vms1-TAP as indicated.
(B) WT, vms1D, cdc48D, and vmsD cdc48D strains bearing plasmids expressing either Vms1 or Vms1-HA and Cdc48, Cdc48-myc, or Cdc48(S565G)-myc were
the indicated antibodies.
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472 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
a ufd1-1 mutant (Figure 7E). This is consistent with the idea that
Vms1 and Ufd1 compete for interaction with Cdc48 and Npl4.
The absence of Vms1 would enable a higher fraction of Cdc48
and Npl4 to associate with Ufd1 and be employed in ERAD.
Based on the nature of the vms1D mutant phenotype and its
association with Cdc48, we reasoned that Vms1 must function
more broadly than just mediating Fzo1 degradation. We there-
fore compared the status of ubiquitinated proteins from wild-
type and vms1D strains at day 1.5 of culture. Cytosolic ubiqui-
tin-protein conjugates were similar in the wild-type and vms1D
mutant strains (Figure 7F). A crude mitochondrial fraction, which
is highly contaminated with ER as indicated by the presence of
the Cue1 protein, has a complicated pattern of ubiquitination,
with some ubiquitin-protein conjugates being elevated in the
vms1D mutant and others being lower (Figure 7F). When that
crude mixture was separated by sucrose-gradient purification
to generate more purified mitochondria, however, the vms1D
mutant showed an increased abundance of a subset of ubiqui-
tin-protein conjugates relative to wild-type (Figure 7F, a replicate
is shown in Figure S7D). This suggests that the vms1D mutant
might have decreased ubiquitin-protein conjugates in other
membrane fractions, particularly the ER. The increased abun-
dance of mitochondrial ubiquitin-protein conjugates in the
vms1D mutant is not uniform. Some conjugates are of equal
abundance, and others are decreased in the vms1D mutant,
consistent with the fact that Cdc48 is important for polyubiquiti-
nation in some contexts (Richly et al., 2005).
We hypothesized that Vms1 recruits Cdc48 and Npl4 to mito-
chondria to engage the ubiquitin-proteasome system in mito-
chondrial protein degradation. One alternative explanation for
the slower protein degradation and increased abundance of
ubiquitin-protein conjugates in vms1D mutant mitochondria is
impaired autophagic degradation of mitochondria or mitophagy
fusion protein. In log phase, a mitochondrially targeted DHFR-
GFP reporter is intact and localized to mitochondria (Okamoto
et al., 2009) (Figure 7G). Upon induction of mitophagy, however,
mitochondria are trafficked to the vacuole and the protein is
partially degraded, leaving a protease-resistant GFP fragment.
At day 2.5 of culture, both the wild-type and vms1D mutant
showed the processed GFP fragment with the vms1D mutant
actually having higher levels, indicative of increased mitophagy.
To address mitophagy in a complementary manner, we deter-
mined the levels of a variety of mitochondrial resident proteins.
When normalized to the cytosolic protein Pgk1, the vms1D
mutant showed elevated Fzo1 as observed previously (Fig-
ure 7H). On the other hand, the mutant showed decreased levels
of the mitochondrial proteins Cox1, Cox2, Porin, and Rip1 (Fig-
ure 7H). The increase in autophagic protein degradation in the
vms1D mutant means that the delayed Fzo1 degradation in the
vms1D mutant is an underrepresentation of the actual impair-
ment in proteasomal degradation of this protein. Mitochondria
from the vms1D mutant show loss of respiratory function and
increased oxidative stressand proteinubiquitination, all ofwhich
would be predicted to induce mitophagy. We therefore hypoth-
esize that the vms1D mutant has impaired ubiquitin-dependent
protein degradation, and one of the indirect effects of that is
If the vms1D mutant has impaired proteasomal degradation of
mitochondrial proteins, it might be more reliant on internal mito-
chondrial proteases, like Oma1 and Yme1, for protein quality
control. The Oma1 protease, which appears to have little func-
tion in unstressed cells, plays a role in mitochondrial integral
inner-membrane protein quality control in yeast (Bestwick
et al., 2010) and is activated upon mitochondrial dysfunction in
mammalian cells (Ehses et al., 2009; Head et al., 2009). Yme1
is the principal subunit of the intermembrane space i-AAA
protease and is responsible for protein degradation in that
compartment (Koppen and Langer, 2007). While the vms1D
single mutant showed a slight growth defect on glycerol and
the oma1D mutant had no glycerol growth defect, the oma1D
vms1D double mutant exhibited a near complete loss of growth
on glycerol (Figure 7I). Under these conditions, there was only
very slight impairment of growth on glucose, indicating that the
phentoype is specific for respirative growth (Figure 7I). A similar
synthetic phenotype was observed between the vms1D muta-
tion and loss of the Yme1 protease (Figure 7J). Together, these
data show that Vms1 is required for normal ubiquitin-proteaso-
mal degradation of mitochondrial proteins. In its absence, the
cell becomes more reliant on alternative protein degradation
strategies, including mitophagy and intrinsic mitochondrial
Herein, we show that Vms1 is part of an evolutionarily conserved
mitochondrial stress-responsive system that promotes mito-
chondrial 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 mitochon-
drial function. By analogy to the ERAD system that responds to
unfolded proteins in the ER, accumulation of damaged, ubiquiti-
nated, 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
Cdc48 and Npl4, thereby promoting ubiquitin-dependent
protein degradation. First, mitochondrial perturbations cause
(C) The vms1D, vms1D npl4D, ufd1D, and vms1D ufd1D strains containing plasmids expressing either Vms1 or Vms1-HA and either Npl4-myc or Ufd1-myc were
grown to late log phase and subjected to immunoprecipitation and immunoblot as in (B).
(D) The ufd1D, ufd1D npl4D, vms1D, and ufd1D vms1D strains containing plasmids expressing either Ufd1 or Ufd1-HA and either Npl4-myc or Vms1-myc were
analyzed as in (B).
(E) The vms1D strain containing plasmids expressing either untagged full-length (?) or HA-tagged full-length (FL), C terminus-only (DN), or N terminus-only (DC)
Vms1 deletion mutants and either untagged or myc-tagged Cdc48 were analyzed as in (B).
(F) The vms1D strain containing plasmids expressing either untagged full-length (?) or HA-tagged full-length (FL), C terminus-only (DN), or N terminus-only (DC)
Vms1 deletion mutants and either untagged or myc-tagged Npl4 were analyzed as in (B).
Mitochondrial Stress and Protein Degradation
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 473
WT + ev
vms1Δ + ev
SD-Ura SD-Ura + Rapamycin
- - + - - +
Lys Sup IP Lys Sup IP Lys Sup IP
Empty vectorVms1-Flag-HA Vms1(VIMΔ)-Flag-HA
1 2 3 4 5 6 7 8 9 Lane :
Figure 5. Vms1 Interacts with Cdc48/Npl4 through a VIM, and this Interaction Is Required for Vms1 Function
(A) Sequence alignment showing the VCP-interacting motif (VIM) from known VCP-interacting proteins and VMS1 orthologs. The amino acids mutated in human
Vms1 for use in (F) are bracketed above the sequence. The amino acids deleted in yeast Vms1 for use in (B)–(D) are bracketed below the sequence.
(B) The cdc48D vms1D strain containing plasmids expressing either Vms1, Vms1-HA, or Vms1 (VIMD)-HA and Cdc48-myc was grown to late log phase and sub-
jected to immunoprecipitation using anti-HA antibody. Immunoblots of crude lysates and immunoprecipitates were developed with the indicated antibodies.
(C) The vms1D npl4D strain containing plasmids expressing either Vms1, Vms1-HA, or Vms1 (VIMD)-HA and Npl4-myc was grown to late log phase and analyzed
as in (B).
(D) WTand vms1Dstrainstransformed withempty vector orcentromericplasmids expressing Vms-HAor Vms1(VIMD)-HA from theendogenous VMS1promoter
were grown to saturation in SD-Ura medium. Five-fold serial dilution of equivalent cell numbers was then spotted on both SD-Ura (left) and SD-Ura + 20 ng/ml
rapamycin (right) plates and grown at 30?C.
(E) C2C12 cells stably expressing either C-terminally Flag-HAtagged VMS1 (+) or empty vector(?) wereharvestedand subjected to two-step affinitypurification:
immunoprecipitation with anti-Flag antibody and eluted with triple-Flag peptide; and immunoprecipitation with anti-HA antibody and eluted with HA peptide.
Equivalent amounts of eluates were separated by SDS-PAGE and subjected to silver staining.
(F) Control C2C12 cells or cells stably expressing either mouse VMS1-Flag-HA or VMS1(VIMD)-Flag-HA wherein VIM residues 684–686 (ERR—as indicated in A)
were mutated to AAA were harvested, and the lysates were subjected to immunoprecipitation with anti-Flag antibody. Lysates (Lys), immunodepleted superna-
tants (Sup), and immunoprecipitates (IP) were analyzed by western blot for VMS1 and VCP. VMS1-Flag-HA and VMS1(VIMD)-Flag-HA were expressed at and
immunoprecipitated in equal amounts (upper band; lower band is endogenous VMS1). The asterisk indicates a nonspecific band. Tubulin was used as a loading
control for lysates and immunodepleted supernatants.
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474 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
WT + ev
vms1Δ + ev
WT + pCDC48
vms1Δ + pCDC48
WT + pNPL4
vms1Δ + pNPL4
vms1Δ + pUFD1
WT + pUFD1
SD-Ura + Rapamycin
DIC mtRFP Cdc48-GFP Merge
SD-Leu-Ura + H2O2
DIC mtRFP Cdc48-GFP Merge
vms1Δ + pVMS1
Ratio of mitochondria co-localized GFP
intensity to total cellular GFP intensity
Figure 6. Vms1 Is Required for the Mitochondrial Translocation of Cdc48
(A) WT and vms1D strains expressing a functional Cdc48-GFP fusion protein from the native CDC48 locus were transformed with mito-RFP plasmid and either
empty vector or centromeric pVMS1-HA and cultured in SD-Leu-Ura at 30?C. Upon reaching mid-log phase, the culture was treated with either vehicle (left) or
3 mM hydrogen peroxide for 3 hr (right) and imaged. Representative images are shown.
(B) Ratio of mitochondria (mito-RFP) colocalized Cdc48-GFP to total cellular Cdc48-GFP signal (± SEM) is graphed for each strain and condition from (A). At least
50 blindly selected cells were analyzed for each strain and condition.
(C) WT and vms1D strains transformed with either pRS426 (ev), pRS426-CDC48, pRS426-NPL4, or pRS426-UFD1 were grown to saturation in SD-Ura medium
and serial 5-fold dilutions were spotted on both SD-Ura (left) and SD-Ura + 20 ng/ml rapamycin (right) plates and grown at 30?C.
Mitochondrial Stress and Protein Degradation
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 475
Day 2.5 cultureWCE
mdm30Δ vms1Δ mdm30Δ
α-Pgk1 α-HA WT Δ WT Δ
WT Δ WT Δ
0 15 30 45 60 75 90 105 120
0 30 60 90 120
Figure 7. Vms1 Is Required for Normal Ubiquitin-Dependent Mitochondrial Protein Degradation
(A) WT, vms1D, mdm30D, and vms1D mdm30D strains were grown in SD complete medium for 2.5 days and whole-cell extracts were prepared and subjected to
immunoblot using anti-Fzo1 antibody, with anti-Pgk1 being a loading control.
(B) WT, vms1D, cdc48-S565G, and vms1D cdc48-S565G strains were treated as in (A).
(C) WT and vms1D strains expressing an HA-tagged allele of Fzo1 were grown to log phase and treated with 0.1 mg/ml cycloheximide. At the times indicated,
samples were harvested and subjected to immunoblotting using anti-HA and anti-porin antibodies. Please note that cycloheximide causes rapid Vms1 translo-
cation to mitochondria. NS indicates a nonspecific band that is present in the absence of the plasmid encoding Fzo1-HA.
(D) WT and vms1D strains expressing CPY*-HA were treated as in (C).
(E) WT and ufd1-1 strains expressing CPY*-HA were treated as in (C).
Mitochondrial Stress and Protein Degradation
476 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
mitochondrial translocation of Vms1 as demonstrated both by
chondria. Each of the experimental conditions under which the
vms1D 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 per-
formed at mitochondria.
Second, Vms1 is required for normal mitochondrial recruit-
ment 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 vms1D 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 vms1D 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 the 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, sug-
gesting 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 copurify
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 copurify in
multiple experimental formats in our hands, and there is no indi-
cation that Vms1 and Ufd1 interact in published data sets.
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 impor-
dation are also distinct. The vms1D mutant has impaired Fzo1
degradation, but under identical conditions has modestly accel-
erated degradation of CPY*, which is almost absent in a ufd1-1
mutant. Genetically, both Cdc48 and Npl4 partially suppressed
the vms1D 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 sub-
strate of the ubiquitin/proteasome system in yeast is the outer
membrane protein Fzo1, and its degradation is impaired in the
vms1D 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 degrada-
tion is not a major cause of the vms1D mutant phenotypes we
have observed. We, therefore, anticipate that the degradation
of other proteins associated with the mitochondrial outer
degradation appears to be widely compromised in the vms1D
mutant as evidenced by the altered accumulation of many poly-
Sixth, loss of Vms1 increases cellular reliance on other, non-
proteasomal modes of mitochondrial protein degradation. We
observed enhanced mitophagy in the vms1D 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 glyc-
erol 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-protea-
some system, participate in the degradation of internal mito-
chondrial proteins? In mammalian cells, the intrinsic mitochon-
drial inner membrane proteins, UCP-2 and UCP-3, have both
(F) WT and vms1D strains were grown in YPD medium for 5 days, harvested, and subjected to mitochondria isolation by differential centrifugation. The crude
mitochondrial fraction (Crude Mito) was then loaded on a sucrose cushion to separate mitochondria (Pure Mito) from other membranes. Equivalent amounts
of proteins were TCA precipitated and immunoblotted using anti-ubiquitin antibody. Anti-Pgk1, Cue1, and Tom20 were used as a marker and loading control
for cytoplasm, ER, and mitochondria, respectively.
(G) WT and vms1D strains transformed with DHFR-GFP were grown in SD-Ura medium either to log phase or for 2.5 days. Equivalent numbers of cells were har-
vested, lysed, and subjected to SDS-PAGE followed by immunoblotting using anti-GFP and anti-Pgk1 (loading control) antibodies. The asterisk indicates
a DHFR-GFP fragment that is targeted to mitochondria and is degraded upon mitophagy, similar to full-length DHFR-GFP.
(H) Two independent cultures of WT and vms1D strains were grown in SD complete medium for 2.5 days and equivalent numbers of cells were harvested, lysed,
and subjected to immunoblot using the indicated antibodies.
(I) WT, vms1D, oma1D, and vms1D oma1D strains were grown in SD complete medium for 1.5 days. Five-fold serial dilution of equivalent numbers of cells were
spotted on both YPAD and YPAGlycerol plates and grown at 30?C.
(J) WT, vms1D, yme1D, and vms1D yme1D strains were analyzed as in (I).
Mitochondrial Stress and Protein Degradation
Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc. 477
recently beenshowntohaveunusually shorthalf-lives, whichare
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 ER in ERAD, might extrude
proteins for degradation in the cytosol.
Finally, the aberrant cellular physiology of the vms1D 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. Concur-
rently, we also observed an increase in oxidative stress and its
damaging effects. Likely as a direct consequence, the vms1D
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 mito-
chondrial protein quality control for mitochondrial function and
healthy life span has been recently emphasized by studies of
the mitochondrial matrix Lon protease (Luce and Osiewacz,
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 mitochon-
that the Vms1-dependent translocation to mitochondria enables
Cdc48 and its cofactor Npl4 to perform a function on mitochon-
dria that is similar to its function in ERAD. In the absence of
late, causing progressive mitochondrial dysfunction and eventu-
ally 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 mitochon-
drial function through protein quality control. In its absence,
progressive mitochondrial dysfunction causes shortened life
span 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 Alzheimer’s 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 alter-
ations 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.
The vms1D strain was transformed with both pVMS1-GFP (or pVMS1 deletion
mutant-GFP) and pMito-RFP plasmids. To test the effect of rapamycin treat-
ment on Vms1 localization, strains were grown to mid-log phase at 30?C in
SD medium lacking both uracil and leucine, treated with vehicle or rapamycin
(200 ng/ml) for 3 hr, and imaged using a Zeiss Axioplan 2 Imaging microscope
(Carl Zeiss). Hydrogen peroxide (3 mM for either 90 min [Vms1 localization] or
3 hr [Cdc48 localization]), antimycin A, oligomycin, CCCP, FCCP (10 mM for
3 hr), and stationary phase experiments were done otherwise identically. For
Cdc48 and Npl4 localization, the WT and vms1D strains expressing C-termi-
nally GFP-tagged Cdc48 or Npl4 from the native CDC48 or NPL4 locus were
treated as above.
manner. The GFP signal intensity that overlapped with mito-RFP (designated
mitochondria) was quantified. Average total cellular GFP signal intensity was
also quantified for each cell. Mitochondrial localization was expressed as
a ratio of mitochondrially colocalized GFP and total cellular GFP.
Yeast Vms1 Tandem Affinity Purification Purification
Tandem affinity purification (TAP) purification was performed as previously
described (Puig et al., 2001). The vms1D strain, transformed with C-terminally
TAP-tagged Vms1 construct under the native VMS1 promoter, was grown to
late log phase and harvested. Cleared lysates were generated and incubated
for 2 hr at 17?C. The TEV cleavage eluate was then incubated with calmodulin
beads for 2 hr at 4?C and the final eluates were obtained with EGTA elution.
Eluates were then analyzed by SDS-PAGE and Coomassie blue staining.
The unique bands detected were identified by LC-MS-MS. For negative
control, JRY472strain transformedwithemptyvector wasanalyzed inparallel.
Fzo1 and CPY* Degradation Assay
WT, vms1D, and ufd1-1 strains transformed with either pRS414-Fzo1-HA or
pRS414-CPY*-HA construct were grown to log phase and treated with
0.1 mg/ml cycloheximide. For each time point, the same number of cells
was harvested, washed, and lysed for each culture as described (Kushnirov,
2000). Each lysate was then subjected to western blotting using anti-HA and
porin antibodies. The levels of Fzo1-HA and CPY*-HA were normalized to
that of porin and Pgk1 in the same sample, respectively. Note that cyclohex-
imide causes Vms1 translocation to mitochondria.
Tandem Affinity Purification and Immunoprecipitation
from Mammalian Cells
C2C12 cells stably expressing an empty integration cassette or mouse Vms1
(NM_026187.4) with a C-terminal Flag-HA tag (ACGGATCCAGCCGCCGACT
TACGCT) weregrownto90%confluency.Cells werelysed inbuffercontaining
40 mM HEPES, 100 mM NaCl, 5 mM Na4P2O7, 5 mM 2-glycerophosphate,
10 mM NaF, 20 mM ZnCl2, 0.02% Igepal 630, and EDTA-free Complete
Protease Inhibitor mix (Roche), at pH 7.5. Lysates were clarified by centrifuga-
tion for 10 min at 16,000 3 g. Vms1-Flag-HA was immunoprecipitated from
supernatants by incubation for 2 hr with agarose beads preconjugated to
either anti-Flag (Sigma, F2426) or anti-HA antibody (Sigma, A2095). For the
TAP of Vms1, the clarified lysates from 4 3 10 cm dishes were pooled and
subjected to immunoprecipitation by Flag, and then washed four times with
wash buffer (lysis buffer but with 120 mM NaCl and 0.1% Igepal-630). Washed
Vms1-Flag-HA was eluted by incubation with 250 mg/ml 33 flag peptide for
45 min on ice and then repurified by HA, washed two times with wash buffer,
and eluted by incubation with 250 mg/ml HA peptide for 45 min at RT. Immu-
noprecipitates were subjected to SDS-PAGE and the resultant gel was silver
stained using the Pierce Silver Stain for Mass Spectrometry (24600). To test
whether an intact VIM was required for coimmunoprecipitation of Vms1-
Flag-HA and p97, complexes were pulled down by Flag and eluted by heating
with 1.53 Laemmli’s buffer. Clarified lysates, supernatants, and eluates were
immunoblotted for Vms1 using an affinity-purified anti-mouse Vms1 antibody
Mitochondrial Stress and Protein Degradation
478 Molecular Cell 40, 465–480, November 12, 2010 ª2010 Elsevier Inc.
that was generated by Cell Signaling Technology by immunizing rabbits. VCP/
p97 antibody from mouse was from Abcam (ab11433).
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
We wish to thank members of the Rutter laboratory as well as David Stillman,
Janet Shaw, Dennis Winge, and Jerry Kaplan and their laboratories for tech-
nical support and helpful discussions. We thank Janet Shaw for the Fzo1-
HA and mito-RFP constructs and fluorescence microscopy; the Stillman lab
for plasmids and yeast strains; the Winge lab for antibodies, protocols, and
oxygen consumption assays; and William Lennarz for the CPY*plasmid. We
also thank members of the Ashrafi laboratory for helpful discussions and tech-
nical assistance, members of the Kenyon lab for help with life span analysis
software, Shohei Mitani and the National Bioresource Project for strains, and
Stefan Taubert for reagents. This work was supported by National Institutes
of Health (NIH) grants DK071962 and GM087346 (to J.R.) DK070149 (to
K.A.), and GM75061 (to J.L.B.); grants from the Israel Science Foundation,
the USA-Israel Binational Science Foundation, and the Israel Cancer Associa-
tion (to M.H.G.); and FWF grant S-9304-B05 and LIPOTOX (to F.M.). E.B.T.
was supported by an American Heart Association Postdoctoral Fellowship
Received: October 22, 2009
Revised: July 21, 2010
Accepted: August 18, 2010
Published: November 11, 2010
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