Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

University of Strasbourg, Strasburg, Alsace, France
Molecular biology of the cell (Impact Factor: 4.47). 08/2012; 23(20):4054-64. DOI: 10.1091/mbc.E12-01-0001
Source: PubMed
ABSTRACT
Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide-dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27(S613A), MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28-green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.

Full-text

Available from: Joëlle Morvan
4054 | J. Morvan et al. Molecular Biology of the Cell
M BoC | ARTICLE
Pkh1/2-dependent phosphorylation of Vps27
regulates ESCRT-I recruitment to endosomes
Joëlle Morvan*, Bruno Rinaldi, and Sylvie Friant
Department of Molecular and Cellular Genetics, Unité Mixte de Recherche 7156, Université de Strasbourg and Centre
National de la Recherche Scientifique, 67084 Strasbourg, France
ABSTRACT Multivesicular endosomes (MVBs) are major sorting platforms for membrane
proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydro-
lase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward
budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and
ILV formation are achieved by the concerted function of endosomal sorting complex re-
quired for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this
pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/
Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phospho-
rylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine
613 and demonstrate that this phosphorylation is required for proper Vps27 function. In-
deed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27
S613A
, MVB
sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the
Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that
Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT com-
plex recruitment at the endosomal membrane.
INTRODUCTION
The multivesicular endosome (or multivesicular body [MVB]) is a
major sorting platform for membrane proteins. The endosomal
membrane undergoes an unconventional inward budding, resulting
in the formation of intraluminal vesicles, and upon fusion between
the MVB and the lysosome/vacuole their content is degraded or
matured by resident hydrolases. MVB membrane invagination and
protein sorting are achieved by the concerted function of four
protein complexes—namely, endosomal sorting complex required
for transport (ESCRT)-0 to ESCRT-III. The ESCRT machinery, first dis-
covered in yeast, is highly conserved throughout evolution and is
well characterized (Saksena et al., 2007; Williams and Urbe, 2007;
Hurley, 2008; Wollert et al., 2009).
The ESCRT-0 complex is composed of vacuolar protein sorting
(Vps) 27/hepatocyte growth factor (EGF)-dependent tyrosine kinase
substrate (Hrs) and Hbp, STAM, EAST (Hse) 1/signal transducing
adaptor molecule (STAM); ESCRT-I of Vps23/tumor suppressor
gene 101 (TSG101), Vps28, Vps37, and Mvb12; ESCRT-II of Vps36,
Vps22, and Vps25; and ESCRT-III of Vps20, Snf7, Vps24, and Vps2
(Teo et al., 2006; Gill et al., 2007; Obita et al., 2007; Im et al., 2009).
The endosomal recruitment of these complexes is sequential and
occurs via direct interaction between subunits of two different com-
plexes. The structure of some of these complexes and of the do-
mains important for cargo recognition reveals the interaction inter-
faces and specific recognition motifs between the complexes
(Hierro et al., 2004; Im et al., 2009; Ren et al., 2009; Boura et al.,
2011). The disassembly of the complexes is achieved by the cata-
lytic activity of the ATPases associated with various cellular activities
(AAA) protein Vps4 and its regulatory subunits Vta1 and Vps60
(Lata et al., 2008).
A key player of these complexes is the ESCRT-0 subunit Vps27/
Hrs. It localizes to endosomal membrane through the interaction of
Monitoring Editor
Judith Klumperman
University Medical Centre
Utrecht
Received: Jan 3, 2012
Revised: Jul 2, 2012
Accepted: Aug 15, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-01-0001) on August 23, 2012.
*Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire,
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104,
Institut National de la Santé et de la Recherche Médicale U 964, 67404 Illkirch
Cedex, France.
Address correspondence to: Joëlle Morvan (morvan@igbmc.fr) or Sylvie Friant
(s.friant@unistra.fr).
© 2012 Morvan et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB
®
,” “The American Society for Cell Biology
®
,” and “Molecular Biology of
the Cell
®
” are registered trademarks of The American Society of Cell Biology.
Abbreviations used: CIP, calf intestinal alkaline phophatase; Cps1, carboxypepti-
dase S; CPY, carboxypeptide Y; ESCRT, endosomal sorting complex required for
transport; HA, hemagglutinin; Hrs, hepatocyte growth factor–dependent tyrosine
kinase substrate; MVB, multivesicular body; Pkh, Pkb-activating kinase homologue;
UEV, ubiquitin E2 variant; Vps, vacuolar protein sorting; Yck, yeast casein kinase;
Ypk, yeast protein kinase.
Page 1
Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4055
Here we show that phosphorylation of
Vps27 is required to regulate ESCRT-0 func-
tion. We establish that Vps27 is phosphory-
lated by the Pkh1/2 kinases on the serine
613 residue. Moreover, we show that this
phosphorylation regulates Vps27 cellular
functions, as it is necessary for proper endo-
somal recruitment of ESCRT-I and MVB sort-
ing of cargoes.
RESULTS
Vps27 is phosphorylated on serine 613
The ESCRT cascade needs to be highly reg-
ulated for proper endosomal-sorting func-
tion. Indeed, in the absence of only one
ESCRT subunit, cells accumulate aberrant
endosomal structures termed class E com-
partment, and sorted proteins are blocked
in this compartment. Vps27 is a key regula-
tor of this MVB-sorting process, and thus its
cellular function most likely requires regula-
tion. Large-scale phosphoproteomic studies
identified several phosphorylation sites in
Vps27 (Gruhler et al., 2005; Smolka et al., 2007; Albuquerque et al.,
2008). Given that phosphorylation/dephosphorylation regulates
many important cellular processes, we speculated that Vps27 func-
tion could also be regulated by phosphorylation.
To analyze the in vivo phosphorylation status of Vps27, we used
a Vps27-hemagglutinin (HA)–tagged protein that rescues the class E
compartment phenotype displayed by the vps27 mutant cells. A
total extract of vps27 cells expressing Vps27-HA was treated with
calf intestinal alkaline phosphatase (CIP; Figure 1A). By comparing
migration with and without CIP treatment, we observed that upon
phosphatase treatment Vps27-HA migrates at a lower molecular
weight (Figure 1A), demonstrating that Vps27-HA is phosphorylated
in vivo. The C-terminal domain of Vps27 (residues 581–622) is re-
quired for ESCRT-I recruitment in vivo (Katzmann et al., 2003). Thus
we analyzed the phosphorylation status of a C-terminal–truncated
version of Vps27 lacking residues 581–622 (vps27
Cter
). We observed
that the migration profile of vps27
Cter
was unchanged after CIP
treatment (Figure 1B). Thus the main Vps27 phosphorylation sites
are located in the C-terminal domain.
Based on phosphoproteomics, only one putative phosphoryla-
tion site–serine 613–lies in the C-terminal region (581–622) of Vps27
(Gruhler et al., 2005; Smolka et al., 2007; Albuquerque et al., 2008).
Thus we generated the vps27
S613A
mutant by site-directed muta-
genesis and analyzed its phosphorylation profile. Total extract of
vps27 transformed by plasmid encoding vps27
S613A
-HA was
treated with CIP (Figure 1C). In contrast to the difference observed
for wild-type Vps27, the migration profile of vps27
S613A
-HA was not
changed after CIP treatment. We also generated point mutants
(S155,157A; S274A; S279,280A; S495A; and T497A) for each of the
other phosphoresidues identified in Vps27. None of them displayed
an altered steady-state phosphorylation profile (Supplemental Fig-
ure S1), demonstrating that the phosphorylation of Vps27 occurs
predominantly on the S613 residue. Next we generated the phos-
phomimetic mutant of Vps27, vps27
S613D
-HA and compared its mi-
gration profile with that of Vps27-HA and vps27
S613A
-HA in vps27
(Figure 1D). We observed that vps27
S613D
-HA migrated at the same
apparent molecular weight as the wild-type protein, whereas the
vps27
S613A
-HA migrated faster. Furthermore, we observed two
bands corresponding to Vps27-HA, a more abundant, upper band
its Fab1/YOTB1/Vac1/EEA1 (FYVE) domain with phosphatidylinosi-
tol 3-phosphate. Vps27/Hrs binds the ubiquitin present on cargoes
to be sorted via its ubiquitin-interacting motif (UIM), and it recruits
the other complexes to endosomes by direct interaction with the
ESCRT-I subunit Vps23/TSG101 (Hurley, 2008). This interaction oc-
curs between the PSDP motifs of Vps27 and the ubiquitin E2 variant
(UEV) domain of Vps23 (Pornillos et al., 2002; Katzmann et al., 2003;
Ren and Hurley, 2011).
ESCRT-I does not directly interact with the endosomal mem-
brane, and therefore its specific recruitment to endosomes oc-
curs through interaction with ESCRT-0. However, the temporal
regulation of ESCRT complex endosomal recruitment and of their
association during MVB formation is unclear. Posttranslational
modifications of ESCRT subunits by phosphorylation and ubiq-
uitination were suggested to regulate/coordinate their function.
In mammalian cells a recent study showed that the ESCRT-0 func-
tion is regulated by a kinase interacting with STAM and modulat-
ing its ubiquitination status to favor EGF receptor internalization
into MVBs (Hanafusa et al., 2011). It was also shown that Hrs is
phosphorylated after EGF stimulation, but the role of this modi-
fication is under debate, even though it was suggested to be
associated with Hrs degradation (Urbe et al., 2000; Stern et al.,
2007).
In this study we aim to decipher the role of ESCRT-0 modification
by analyzing yeast Vps27/Hrs phosphorylation and identifying the
involved kinase. In Saccharomyces cerevisiae, only a few kinases in-
volved in the endocytic trafficking pathway have been identified,
among them the conserved Pkh-Ypk kinase cascade (Friant et al.,
2001; deHart et al., 2002). The Pkh1/2 kinases, the homologues of
mammalian 3-phosphoinositide–dependent kinase, are activated
by sphingoid bases and phosphorylate the Ypk1 serine–threonine
kinase. Ypk1, the homologue of mammalian serum and glucocorti-
coid–induced kinase, is required for endocytosis but is not neces-
sary for receptor phosphorylation or ubiquitination (deHart et al.,
2002). This receptor modification is instead devoted to the yeast
casein kinase homologues Yck1 and Yck2, which phosphorylate
plasma-membrane proteins, allowing their subsequent ubiquitina-
tion and internalization (Hicke et al., 1998; Feng and Davis, 2000;
Marchal et al., 2000).
FIGURE 1: Vps27 is a phosphoprotein and is phosphorylated on residue S613 in the C-terminal
domain. vps27 cells transformed with pRS426-Vps27-HA (A), YCpHAC33-vps27
Cter
-HA (B), or
YCpHAC33-Vps27 and YCpHAC33-vps27
S613A
-HA (C) total extracts were treated with (+) or
without (–) CIP. Samples were analyzed by SDS–PAGE, followed by Western blot using anti-HA
antibodies. NT, nontreated. (D) Total extracts of vps4 or vps27 cells transformed with pRS426-
Vps27-HA, pRS426-vps27
S613A
-HA, and pRS426-vps27
S613D
-HA were analyzed by SDS–PAGE,
followed by Western blot using anti-HA antibodies.
Page 2
4056 | J. Morvan et al. Molecular Biology of the Cell
ypk2 mutant strains. This CPY missorting
displayed by the pkh2 pkh1-ts strain was
more pronounced at 30 than at 25°C and
was not restricted to CPY, since on acidic
milk plates a clear halo of digestion due to
vacuolar hydrolase secretion was also ob-
served (Supplemental Figure S2). Moreover,
this mistargeting of vacuolar hydrolases was
rescued by the reintroduction of wild-type
Pkh2-HA in the pkh2 pkh1-ts mutant and
to a lesser extent by the kinase-defective
mutant pkh2
K208R
(Supplemental Figure S2).
Of interest, the kinase-inactive pkh2
K208R
mutant did not recapitulate the strong vps
phenotype displayed by the pkh-ts cells,
suggesting either a partially kinase-inde-
pendent requirement for this Pkh2 protein
or that this kinase mutant is not fully inactive
(Inagaki et al., 1999). The pkh2
K208R
mutant
is considered as kinase inactive; however, in
the initial description and analysis of this mutant, the in vitro phos-
phorylation assay shows that this mutant had much lower kinase
activity than the corresponding wild-type Pkh2 but was not fully in-
active (Inagaki et al., 1999). Our results suggest that the Pkh1/2-
Ypk1 kinase cascade regulates the VPS pathway.
Because the pkh2 pkh1-ts and ypk1 strains displayed a strong
CPY secretion phenotype even at the permissive temperature of
30°C, we analyzed Vps27-HA phosphorylation status in these strains.
Total extracts from wild-type, pkh2, pkh2 pkh1-ts, ypk1, and
ypk2 strains transformed with a plasmid encoding for Vps27-HA
and grown at 30°C were analyzed (Figure 2B). In pkh2, ypk1, and
ypk2 mutant cells, as in wild-type cells, Vps27-HA was present as a
doublet, with the more abundant, upper band corresponding to the
phosphorylated form of Vps27-HA. In contrast, in the pkh2 pkh1-ts
strain, the upper band signal was strongly decreased compared with
that of the lower band, indicating that Vps27-HA phosphorylation
was impaired in this mutant. This result demonstrates that Pkh1/2
are required for proper steady-state phosphorylation of Vps27 in
vivo.
Pkh2 phosphorylates Vps27 at the S613 residue in vitro
To investigate whether Pkh2 directly phosphorylates Vps27, we per-
formed an in vitro phosphorylation assay. Purified recombinant
hexahistidine (6xHis)-Vps27 produced in Escherichia coli thus made
void of posttranslational modifications was incubated with Pkh2-HA
or kinase-inactive pkh2
K208R
-HA immunoisolated from pkh1 cells
(Supplemental Figure S3) in the presence of [γ-
32
P]ATP. The Pkh2-HA
construct was active, as it rescued the temperature-sensitive growth
and the vacuolar hydrolases secretion of the pkh2 pkh1-ts strain,
and a similar complementation was observed for the kinase-inactive
pkh2
K208R
-HA construct, albeit to a lesser extent than for the wild-
type Pkh2 (Supplemental Figure S2).
We observed a weak band corresponding to
32
P-labeled 6xHis-
Vps27 in the absence of Pkh2-HA (Figure 3A, lane 2). On addition of
Pkh2-HA beads to the phosphorylation mix a strong signal for the
high–molecular weight band corresponding to Pkh2 autophospho-
rylation was observed (Figure 3A, lane 3). This shows that the kinase
is active in our in vitro assay. Incubation of the Pkh2-HA kinase with
6xHis-Vps27 induced Vps27 phosphorylation (Figure 3A, lane 4),
compared with the weaker signal observed in absence of the kinase
(Figure 3A, lane 2). In contrast, in the presence of the kinase-inactive
mutant pkh2
K208R
(Figure 3A, lane 6), the signal corresponding to
corresponding to the phosphorylated form and a second, minor,
lower band corresponding to the unphosphorylated form. This
shows that at steady state Vps27 is mainly present in a phosphory-
lated form. Moreover, a similar profile was observed for Vps27-HA
and vps27
S613A
-HA in the vps4 mutant defective in the disassembly
of the ESCRT proteins (Figure 1D), showing that Vps27 is phospho-
rylated in class E mutant cells. These results indicate that the C-ter-
minal phosphorylation of Vps27 occurs mainly at the S613 residue.
The Pkh kinases are involved in Vps27 phosphorylation
To identify the kinase responsible for Vps27 phosphorylation, we
analyzed the kinases required for endocytosis, as MVB sorting and
endocytic internalization share common features, such as ubiquit-
ination of the cargo (Lauwers et al., 2010). Only few kinases are in-
volved in endocytosis. Yeast casein kinase I redundant isoforms Yck1
and Yck2 trigger the ubiquitination and internalization of the α-
factor receptor Ste2 and of the uracil permease Fur4 (Hicke et al.,
1998; Marchal et al., 1998, 2000). Pkh1 and Pkh2, two redundant
kinases, act together with their downstream kinases Ypk1 and Ypk2
and are required for the internalization of Ste2 (Friant et al., 2001;
deHart et al., 2002).
We hypothesized that posttranslational modifications regulate
Vps27 cellular functions, and thus the involved kinase mutant should
display similar phenotypes as vps27 cells. Like all class E vps mu-
tants, vps27 cells mistarget vacuolar hydrolases in the extracellular
medium and secrete carboxypeptidase Y (CPY). To analyze the re-
quirement for the redundant kinases in the VPS pathway, we used
the temperature-sensitive (ts) yck1 yck2-1 and the pkh2 pkh1-ts
strains deleted for one isoform and bearing a temperature-sensitive
allele of the other isoform (Hicke et al., 1998; Marchal et al., 1998;
Friant et al., 2001). The Ypk1 and Ypk2 kinases have redundant es-
sential cellular functions (Chen et al., 1993), but for endocytosis only
the ypk1 cells have an α-factor internalization defect (deHart et al.,
2002), and only Ypk1 is phosphorylated and activated by the Pkh
kinases (Casamayor et al., 1999). We analyzed the function of the
VPS pathway by comparing the CPY secretion displayed by the
yck1 yck2-1, pkh2 pkh1-ts, and ypk1 ypk2 mutants at 25 and
30°C and used the single mutants yck2, pkh1, and pkh2 as con-
trols (Figure 2A). As expected, the wild-type strain did not secrete
CPY, in contrast to the vps27 mutant. At the permissive tempera-
ture of 30°C, pkh2 pkh1-ts and ypk1 strains secreted CPY into the
extracellular medium, in contrast to the yck1 yck2-ts, pkh1, and
FIGURE 2: The pkh2 pkh1-ts strain displays a defect in CPY sorting and in Vps27
phosphorylation. (A) Wild-type (BY4742), vps27, pkh1, pkh2, pkh2 pkh1-ts, WT (LRB341),
yck1, yck1 yck2-ts ypk2, and ypk1 strains were tested for CPY secretion. A 5-μl amount of
culture at OD
600
of 0.4 was spotted on YPD plate and covered with a nitrocellulose membrane.
After 48 h of growth at 30°C, CPY was detected using anti-CPY antibodies. (B) Total cell extracts
of wild-type, pkh2, pkh2 pkh1-ts, ypk1, and ypk2 strains transformed by pRS426-VPS27-
HA were analyzed by SDS–PAGE, followed by Western blot using anti-HA antibodies. Two
individual clones were analyzed (cl1 and cl2).
Page 3
Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4057
phosphorylated 6xHis-Vps27 was similar to the background level
(Figure 3A lane 2). In addition, no autophosphorylation of this
pkh2
K208R
kinase-inactive mutant was detected, ensuring that the
mutation altered the kinase activity of the protein. These results
demonstrate that Pkh2 directly phosphorylates Vps27 in vitro.
We next asked whether the S613 residue was the target of Vps27
phosphorylation by Pkh2. We purified the 6xHis-vps27
S613A
recom-
binant protein from E. coli (Supplemental Figure S3A). 6xHis-tagged
Vps27 and vps27
S613A
were subjected to in vitro phosphorylation by
Pkh2-HA or pkh2
K208R
-HA immunoprecipitated from pkh1 cells
(Figure 3B). In contrast to Vps27, which was phosphorylated by
Pkh2, vps27
S613A
phosphorylation strongly decreased. In the pres-
ence of the kinase-inactive pkh2
K208R
, the residual phosphorylation
observed for Vps27 also decreased for vps27
S613A
(Figure 3B). This
result demonstrates that the S613 residue is specifically required for
Pkh2-dependent phosphorylation of Vps27 in vitro.
Vps27 phosphorylation is required for cargo sorting
at the MVB
Vps27 fails to be properly phosphorylated in pkh2 pkh1-ts cells,
and thus we investigated whether this affected its MVB-sorting func-
tion. We analyzed the trafficking of the carboxypeptidase S Cps1–
green fluorescent protein (GFP), which is normally sorted in a ubiq-
uitin-dependent manner into MVB intralumenal vesicles and then
delivered to the vacuolar lumen (Reggiori and Pelham, 2001). Wild-
type, pkh2, and pkh2 pkh1-ts cells transformed with a plasmid
encoding Cps1-GFP were grown at 30°C and observed for fluores-
cence. In wild-type and pkh2 cells, Cps1-GFP was localized to the
vacuolar lumen (Figure 4A). In contrast, in pkh2 pkh1-ts cells Cps1-
GFP was mislocalized at the vacuolar mem-
brane, showing that the Pkh1/2 kinases are
required for proper MVB sorting.
Because Pkh1/2 kinases phosphorylate
Vps27 and are required for endosomal sort-
ing, we addressed the subcellular localiza-
tion of Pkh2. We used wild-type and class E
vps4 mutant cells bearing PKH2 tagged at
the locus with GFP and observed the local-
ization of Pkh2-GFP by fluorescence micros-
copy (Supplemental Figure S4A). In both
strains, Pkh2-GFP localized mainly to punc-
tae near the plasma membrane and in one
or few intracellular dots that did not colo-
calize with the class E compartment in
vps4 cells. This shows that the Pkh kinases
are not clustered in the aberrant class E en-
dosomal compartment. We also analyzed
the subcellular distribution of Pkh2-GFP in
wild-type and vps4 strains (Supplemental
Figure S4B). Whole-cell lysates (T) were
subjected to differential centrifugation to
generate two membrane fractions—the
13,000 × g pellet (P13) and the 100,000 × g
pellet (P100)—and a cytosolic fraction—the
100,000 × g supernatant (S100). The proper
fractionation was attested by the presence
in P13 and P100 of the soluble N-ethylma-
leimide–sensitive factor attachment protein
receptor Vti1 that cycles between Golgi
and endosomal membranes, in P13 of
the vacuolar membrane alkaline phos-
phatase (ALP), and in S100 of the cytosolic
FIGURE 3: Pkh2 phosphorylates directly Vps27 on residue S613 in
vitro. Anti-HA immunoprecipitation was performed from total lysate of
pkh1 cells transformed with empty plasmid or wild-type or kinase-
inactive (K208R) Pkh2-HA–encoding plasmid. Beads were used in an in
vitro phosphorylation assay with 25 μg of recombinant Vps27-6xHis
(A, B) or vps27
S613A
-6xHis (B) in the presence of [γ-
32
P]ATP. Samples
were analyzed by SDS–PAGE, and the gels were dried and exposed
for 24 h on a phosphoscreen. A representative experiment is shown;
the experiment was repeated at least three times.
FIGURE 4: Pkh1/2 kinases are required for MVB sorting. (A) Wild-type, pkh2, and pkh2
pkh1-ts strains transformed with Cps1-GFP (pGO45) were grown at 30°C until exponential
phase and observed by fluorescence microscopy. (B) pkh2 pkh1-ts cells cotransformed with
Cps1-GFP (pGO47) and empty plasmid (YCpHAC33), YCpHAC33-Vps27-HA, YCpHAC33-
vps27
Cter
-HA, YCpHAC33-vps27
S613A
-HA, or YCpHAC33-vps27
S613D
-HA were grown at 30°C
until exponential phase and observed by fluorescence microscopy.
Page 4
4058 | J. Morvan et al. Molecular Biology of the Cell
vps27
S613D
-HA mutant, Cps1-GFP was prop-
erly localized to the vacuolar lumen. We also
analyzed the vacuolar delivery of the α-factor
receptor Ste2, which follows the endocytic
pathway and is internalized in the intralumi-
nal vesicles of MVBs before its delivery and
degradation into the vacuolar lumen (Stefan
and Blumer, 1999). In the vps27 cells bear-
ing the empty vector or the vps27
Cter
con-
struct, Ste2-GFP accumulated in the class E
compartment, and this accumulation was
also observed for the vps27
S613A
mutant but
to a lesser extent, whereas in the presence of
wild-type Vps27 or of the phosphomimetic
vps27
S613D
mutant, Ste2-GFP was properly
delivered into the lumen of the vacuole
(Figure 5). These results show that the phos-
phorylation of Vps27 on S613 is required for
proper MVB sorting of cargo proteins.
It is striking that the phosphomimetic
vps27
S613D
mutant complemented the CPY
secretion defect displayed by the vps27
cells, whereas the vps27
S613A
mutant showed
only a partial rescue (Supplemental Figure
S2C). We thus wondered whether the phos-
phomimetic vps27
S613D
mutant could com-
plement the VPS defect displayed by the
pkh2 pkh1-ts cells. We analyzed the MVB
sorting of Cps1-GFP in the pkh2 pkh1-ts
cells bearing the empty vector, the Vps27
WT or the vps27
S613A
or vps27
S613D
mutant
at 30°C (Figure 4B). None of the Vps27 vari-
ants rescued the Cps1-GFP mislocalization
at the vacuolar membrane in the pkh2
pkh1-ts cells. We also observed that neither
the temperature sensitivity nor the secretion
of CPY displayed by the pkh2 pkh1-ts cells
were rescued by the expression of Vps27,
vps27
S613A
, or vps27
S613D
(Supplemental Fig-
ure S2C). These results suggest that Vps27
is not the only effector of the Pkh kinase in-
volved in MVB sorting.
To rule out that Vps27 mutants are not
properly localized, we subjected vps27 cells transformed with
empty plasmid or plasmid encoding HA-tagged Vps27, vps27
S613A
,
or vps27
S613D
to subcellular fractionation (Supplemental Figure S5A).
Whole-cell lysates (T) were subjected to differential centrifugation to
generate the P13 and P100 membrane fractions and the S100 cyto-
solic fraction. Equal volumes of the fractions were analyzed by West-
ern blot using anti-HA antibody and antibodies directed against
control proteins (Vps10 and Pgk1). The proper fractionation was at-
tested by the presence of the Vps10 transmembrane receptor, which
cycles between Golgi and endosomes in P13 and P100 and of Pgk1
in S100. Wild-type, S613A, and S613D Vps27 were all found mostly
in the P13 and P100 fractions and to a lesser extent in the S100 frac-
tion. Thus mutation of the S613 residue does not alter Vps27 mem-
brane localization.
Vps27 phosphorylation is required for Vps28-GFP
recruitment to endosomes
We hypothesized that Pkh2-dependent phosphorylation of Vps27
in the C-terminal domain might regulate the interaction with
phospho–glycerate kinase Pgk1 (Supplemental Figure S4B). Pkh2-
GFP was mainly detected in membrane fractions (P13 and P100) in
both wild-type and vps4 cells. These data show that Pkh2-GFP is
associated with plasma membrane and intracellular membranes
but is not enriched in endosomes. We also performed a subcellular
fractionation of Pkh2-HA in wild-type cells and observed the same
distribution as with Pkh2-GFP (Supplemental Figure S4B), demon-
strating that the overproduction or the nature of the tag did not
change the subcellular localization of the protein.
To decipher the role of the S613 residue in the MVB-sorting func-
tion of Vps27, we analyzed the localization of the vacuolar protein
Cps1-GFP in vps27 cells transformed with empty plasmid or plas-
mid encoding HA-tagged Vps27, vps27
S613A
, or vps27
S613D
(Figure 5).
As expected, in the vps27 cells transformed with empty plasmid
and HA-tagged vps27
Cter
, Cps1-GFP was retained in the class E
compartment and at the vacuolar membrane. In vps27 cells ex-
pressing vps27
S613A
-HA, Cps1-GFP was missorted at the vacuolar
membrane but was also found in the vacuolar lumen. In contrast, in
vps27 cells expressing wild-type Vps27-HA or the phosphomimetic
FIGURE 5: Vps27 phosphorylation on Ser613 is required for proper MVB sorting. The vps27
cells cotransformed with Cps1-GFP (pGO47) or Ste2-GFP and empty plasmid (YCpHAC33),
YCpHAC33-Vps27-HA, YCpHAC33-vps27
Cter
-HA, YCpHAC33-vps27
S613A
-HA, or YCpHAC33-
vps27
S613D
-HA were grown at 30°C until exponential phase and observed by fluorescence
microscopy.
Page 5
Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4059
To ensure that this mislocalization of Vps28-
GFP was not due to a mislocalization of
Vps27, we also analyzed the localization of
Vps27 tagged at the locus with GFP in the
wild-type, pkh2, and pkh2 pkh1-ts cells
(Supplemental Figure S6). Vps27-GFP was
detected as large punctae juxtaposed to
the vacuole and corresponding to endo-
somes in all these strains. These results
strongly suggest that Pkh1/2 kinases regu-
late ESCRT-I recruitment to endosomes.
Next we investigated whether the phos-
phorylation of Vps27 at residue S613 was
required for the recruitment of Vps28 to en-
dosomes. We thus analyzed the localization
of chromosomally tagged Vps28-GFP in
vps27 cells transformed with empty plas-
mid or plasmid encoding HA-tagged Vps27,
vps27
Cter
, vps27
S613A
, or vps27
S613D
(Figure
7). As expected, in vps27 cells transformed
with the empty plasmid, GFP-Vps28 was cy-
tosolic. A normal localization at the endo-
somes was restored in the presence of
Vps27-HA, showing that Vps27-HA effi-
ciently recruits ESCRT-I. As previously de-
scribed (Katzmann et al., 2003), we observed
that the vps27
Cter
-HA construct did not al-
low the recruitment of Vps28-GFP at the
endosomes. In vps27 cells transformed
with vps27
S613A
-HA, Vps28-GFP displayed
also mostly a cytoplasmic localization (80%
of cells); in contrast, the phosphomimetic
mutant vps27
S613D
-HA exhibited increased
endosomal localization of Vps28-GFP, even if this rescue was not as
efficient as the one observed for the wild-type Vps27 (Figure 7B). It
was previously shown that in the vps4 class E mutant cells impaired
in ESCRT dissociation from endosomes, the vps27
Cter
mutant was
not defective for Vps23 recruitment on endosomes (Katzmann et al.,
2003). Therefore, we also analyzed the endosomal recruitment of
Vps28-GFP in the vps4 vps27 cells expressing Vps27, vps27
S613A
,
or vps27
S613D
(Supplemental Figure S7). In the vps4 class E mutant
cells, Vps28-GFP endosomal recruitment was restored for vps27
S613A
mutant and ameliorated for the vps27
S613D
mutant. This confirmed
that the S613 residue was not required for the interaction of Vps27
with Vps23. These results demonstrate that the S613 residue is re-
quired for the proper recruitment of Vps28-GFP to endosomes.
Taken together, these results show that phosphorylation of the resi-
due S613 of Vps27 and the presence of Pkh1/2 kinases are required
for the endosomal recruitment of the ESCRT-I complex. We propose
that the phosphorylation status of Vps27 regulates its ESCRT-I re-
cruitment function and thus the coordinated function of the ESCRT
complexes.
DISCUSSION
In this study we report that Vps27 is mainly found in a phosphory-
lated form in vivo and that this phosphorylation occurs in the C-ter-
minal domain of the protein. By screening kinases involved in endo-
cytosis for a general defect in the VPS pathway, we identified Pkh1/2
as the kinases phosphorylating Vps27. In addition, we demonstrated
that Pkh1/2 phosphorylates Vps27 in vivo and in vitro. We identified
the serine 613 as critical for proper phosphorylation of Vps27 in vivo
and as the target of Pkh2 phosphorylation in vitro. Moreover, Vps27
ESCRT-I and thus its recruitment to endosomes. The ESCRT-I com-
plex, composed of Vps23, Vps28, Vps37, and Mvb12, assembles in
the cytoplasm and is recruited to the endosomal membrane via direct
binding between Vps27 and Vps23 (Pornillos et al., 2002; Katzmann
et al., 2003; Ren and Hurley, 2011). Thus we analyzed whether the
phosphorylation of Vps27 on the S613 residue was required for its
direct interaction with Vps23. Pull-down studies revealed that Vps23-
GFP interacted to the same extent with wild-type, S613A, or S613D
Vps27-6His recombinant proteins immobilized on nickel-nitriloacetic
acid beads (Supplemental Figure S5B). We confirmed these results
by performing coimmunoprecipitation of HA-tagged Vps27,
vps27
S613A
, and vps27
S613D
with Vps23-GFP from vps27 cells (Sup-
plemental Figure S5C). Vps23-GFP was able to coimmunorecipitate
Vps27, as well as the vps27
S613A
and vps27
S613D
variants. Thus the
Cps1-sorting defect displayed by cells expressing the vps27
S613A
mutant is not due to a lack of interaction with Vps23. It was previ-
ously shown that Vps27 lacking its C-terminal domain (581–622) is
able to interact with Vps23 (Bilodeau et al., 2003) but is defective for
Cps1 MVB sorting and fails to recruit the ESCRT-I subunit Vps23 on
endosomes (Katzmann et al., 2003). Therefore, we analyzed the en-
dosomal recruitment of the ESCRT-I subunit Vps28-GFP in wild-type,
pkh2, and pkh2 pkh1-ts cells (Figure 6). The GFP tag did not in-
duce Vps28 misfunction, since the cells showed normal vacuoles
and no class E phenotypes (unpublished data). In the wild-type pa-
rental strain as well as in the pkh2 cells, Vps28-GFP localized to a
large punctuate structure juxtaposed to the vacuole corresponding
to the endosomes. In contrast, in pkh2 pkh1-ts cells at permissive
temperature (30°C), Vps28-GFP was mainly localized to the cyto-
plasm as a diffuse fluorescence in most of the cells (77%; Figure 6).
FIGURE 6: Vps28-GFP is not properly recruited to endosomes in the pkh2 pkh1-ts mutant.
(A) Wild-type, pkh2, and pkh2 pkh1-ts strains bearing GFP-tagged Vps28 under its own
promoter were grown until exponential phase of growth at 25°C. Cells were then imaged by
fluorescence microscopy. (B) Quantification was made on two independent clones and >200 cells.
Page 6
4060 | J. Morvan et al. Molecular Biology of the Cell
its anchorage at the plasma membrane (Roth
et al., 2011). This plasma-membrane specific
localization of Yck2 might explain why it can-
not act directly at later stage of endocytosis.
In contrast, we showed that the pkh2 pkh1-
ts mutant displays a strong secretion of vac-
uolar hydrolases, attesting to a general VPS
pathway defect (Figure 2 and Supplemental
Figure S2) and that this defect was not re-
stored by the phosphomimetic vps27
S613D
mutant (Supplemental Figure S2 and Figure
4B), suggesting that Vps27 was not the only
Pkh1/2 effector. Furthermore, we showed
that ypk1 mutant displays a CPY secretion
phenotype (Figure 2), suggesting that the
Pkh1/2 kinases could also act on its down-
stream effector Ypk1 to regulate the vacuolar
sorting of hydrolases. The Pkh1/2 kinases
might also act as a protein platform to regu-
late the trafficking function of some addi-
tional effector(s) via protein–protein interac-
tions, as the pkh2
K208R
mutant that is
considered as kinase inactive did not reca-
pitulate the strong VPS defect displayed by
the pkh2 pkh1-ts mutant cells.
Vps27 binds directly to the UEV domain
of the ESCRT-I subunit Vps23 and recruits
the whole complex to endosomes. It was
shown by pull-down experiments with re-
combinant GST fusion of a truncated ver-
sion of Vps27 that residues 431–485, includ-
ing the
447
PSDP
450
-1 and
523
PSDP
536
-2 motifs
of Vps27, were required for interaction with
the UEV domain of Vps23 (Bilodeau et al.,
2003). However, Katzmann et al. (2003)
showed that the residues 581–622 and the
581
PTVP
584
motif of Vps27 control Vps23 en-
dosomal localization in vivo. Of interest, re-
cent interaction and structural studies con-
firmed the interaction between the PDSP
motifs of Vps27 and an N-terminal motif on
the UEV domain of Vps23 (Ren and Hurley,
2011). However, this interaction motif was not essential for the MVB
sorting of Cps1 (Ren and Hurley, 2011). Here we show that the S613
residue in the C-terminal domain of Vps27 (581–622) is required to
trigger the recruitment of the whole ESCRT-I complex to endo-
somes, as Vps28-GFP was mislocalized in its absence. Moreover, the
single mutation of the phosphorylation site S613 is sufficient to alter
the recruitment of the ESCRT-I complex by Vps27, and this without
altering the capacity of Vps27 to bind to Vps23 (Supplemental Fig-
ure S5, B and C). We hypothesize that the function of this phospho-
rylation is to regulate the recruitment of ESCRT-I, perhaps by facili-
tating the accessibility to the PSDP motifs. In mammalian cells the
Vps27 homologue Hrs is phosphorylated on residue Y334, which is
just upstream of the
348
PSAP
351
motif required for TSG101 interac-
tion, upon EGF stimulation (Urbe et al., 2000; Steen et al., 2002).
These observations allow us to speculate that Hrs–TSG101 interac-
tion might also be modulated by phosphorylation. The role of Hrs
phosphorylation remains unclear; it was suggested that it triggers
relocation of Hrs from cytosol to endosome (Urbe et al., 2000), but
was also described as responsible for Hrs cytosolic relocation and
degradation (Stern et al., 2007). Using subcellular fractionation, we
phosphorylation is required for MVB sorting of cargo like Cps1 and
Ste2, a missorting most likely due to the impaired recruitment
of ESCRT-I to endosomes. Indeed in a pkh2 pkh1-ts mutant, the
ESCRT-I subunit Vps28 is partly mislocalized to the cytoplasm. This
defect is also observed in cells bearing vps27
S613A
as the sole source
of Vps27. Thus Vps27 phosphorylation by Pkh1/2 on serine 613
regulates the recruitment of ESCRT-I to endosomes.
In S. cerevisiae endocytosis is dependent on sphingoid base
(Zanolari et al., 2000). It was shown that Pkh1/2 kinases are activated
by sphingoid base and are required for the internalization step of
endocytosis (Friant et al., 2001). The overexpression of these kinases
can restore endocytosis in the lcb1-100 mutant deficient in sphingo-
lipid synthesis. Here we show that in addition to its role in the early
step of endocytosis, Pkh2 also displays a general defect in the VPS
pathway and can directly phosphorylate Vps27 to regulate ESCRT-I
recruitment to endosomes. Thus Pkh1/2 kinases also play an impor-
tant role at a later stage of endocytosis. Consistent with the observa-
tion of Marchal et al. (2001), we showed that the yck1 yck2-ts mu-
tant did not display a general VPS pathway defect (Figure 2). Of
interest, it was recently shown that Yck2 is palmitoylated, leading to
FIGURE 7: Vps28-GFP is not properly recruited to endosomes in cells producing Vps27 lacking
its C-terminal domain or bearing the S613A mutation. (A) The vps27 vps28-GFP strain was
transformed with empty plasmid (YCpHAC33) or YCpHAC33 plasmid encoding wild-type, Cter,
S613A, or S613D Vps27-HA. Cells were grown until exponential phase of growth at 25°C and
imaged by fluorescence microscopy. (B) Quantification was made on two independent clones
and >200 cells.
Page 7
Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4061
observed no major differences in Vps27 distribution upon mutation
of the S613 residue, suggesting that the phosphorylation of this resi-
due does not alter Vps27 endosomal localization (Supplemental
Figure S5A). An explanation for the importance of Hrs phosphoryla-
tion in ESCRT-I recruitment to endosomes is that phosphorylation of
a residue lying next to the P(T/S)AP motifs might trigger a conforma-
tional change resulting in better accessibility of this motif to ESCRT-I
(TSG101/Vps23) binding.
Several other residues of Vps27 can be phosphorylated (Gruhler
et al., 2005; Smolka et al., 2007; Albuquerque et al., 2008). We ana-
lyzed the steady-state phosphorylation status of point mutants of
these residues (S155,157A; S274A; S279,280A; S495A; and T497A)
and found none required for steady-state phosphorylation of Vps27
(Supplemental Figure S1). However, this does not exclude a role in
regulating Vps27 cellular functions and thus the MVB pathway in
different environmental or stress conditions. Indeed, two of these
residues, S157 and S495, in the FYVE and C-terminal region, re-
spectively, are hyperphosphorylated upon α-factor treatment
(Gruhler et al., 2005). It will be interesting to further investigate the
role of the phosphorylation of these residues and to identify the ki-
nases involved in these modifications.
In the ubiquitin-binding domain containing protein, ubiquitina-
tion is involved in intramolecular inhibition of the ubiquitin binding
(Hoeller et al., 2006). Phosphorylation can regulate ubiquitination of
proteins; for example, in the case of the uracil permease Fur4 phos-
phorylation of the PEST sequence is required for proper ubiquitina-
tion and subsequent internalization of the protein (Marchal et al.,
2000). Thus phosphorylation could also regulate the ubiquitination
of Vps27 and thus its ability to bind ubiquitinated cargoes. It has
been shown that the ESCRT-0 protein STAM (Hse1 in yeast) ubiquit-
ination is increased upon down-regulation of the scaffold leucine-
rich repeat kinase LRRK1, and this regulates endosomal trafficking
of the EGF receptor (Hanafusa et al., 2011). Thus a similar mecha-
nism could exist for the regulation of Vps27 ubiquitin binding via its
UIM motif. It will be important to further characterize the role of the
phosphorylation of the different residues of Vps27.
Phosphorylation of Vps27 at residue S613 is important for the
regulation of ESCRT function, and thus the dephosphorylation step
must also play an important role in this process. One protein phos-
phatase candidate is the phosphatase 2A (PP2A). Indeed, Friant
et al. (2000) showed that its loss of activity, as well as Pkc1 overex-
pression, can suppress sphingoid base requirement for endocytosis.
This suggests that PP2A acts in the same pathway as Pkh1/2 to reg-
ulate endocytosis. Further investigations should be carried out to
decipher the role of this phosphatase in the regulation of MVB
function.
In conclusion, we showed that phosphorylation of Vps27 is cru-
cial for endosomal recruitment of the ESCRT machinery and thus
required for proper MVB sorting, which gives new insight into the
regulation of the ESCRT machinery assembly. Considering the high
degree of conservation of the MVB pathway throughout evolution,
we propose that this might be a conserved regulatory mechanism of
MVB function.
MATERIALS AND METHODS
Strains, plasmids, media, and growth conditions
Yeast strains and plasmids used in this study are listed in Tables 1
and 2, respectively. Yeast strains were transformed using a modified
version of the lithium acetate method (Gietz et al., 1992).
Cells were grown at 30°C or at the indicated temperature in
rich medium (YPD; 1% yeast extract, 2% peptone, 2% glucose) or
synthetic medium (SC; 0.67% yeast nitrogen base without amino
Name Genotype Origin
BY4742
Mat α his31 leu20 lys20
ura30
EUROSCARF
VPS27 Mat α his31 leu20 lys20
ura30 vps27::KanMX4
EUROSCARF
SFY105
Mat α his31 leu20 lys20
ura30 trp1::hphMX4
vps27::KanMX4
This study
YPK1 Mat α his31 leu20 lys20
ura30 ypk1::KanMX4
EUROSCARF
YPK2 Mat α his31 leu20 lys20
ura30 ypk2::KanMX4
EUROSCARF
LRB341
Mat α his3 leu2 ura3-52
Robinson
et al. (1993)
LRB343
Mat α his3 leu2 ura3-52 yck2-
1::HIS3
Robinson
et al. (1993)
LRB346
Mat α his3 leu2 ura3-52 yck1
yck2-1
Robinson
et al. (1993)
INA17-4D Mat a ura3 trp1 leu2 his2 ade1 Inagaki et al.
(1999)
RH5413 Mat a ura3-52 trp1-1 leu2-3112
his2 ade2 lys2 bar1::URA3 pkh1::
TRP1
Friant et al.
(2001)
RH5388 Mat a ura3 trp1 leu2 his2 ade2
pkh2::LEU2
Friant et al.
(2001)
INA106-3D
Mat α ura3 trp1 leu2 his2 ade1
pkh2::LEU2 pkh1-ts
Inagaki et al.
(1999)
SFY104 Mat a ura3 leu2 his2 This study
SFY103 Mat a ura3 his pkh2::LEU2 This study
SFY102
Mat α ura3 his met15 pkh2::LEU2
pkh1-ts
This study
SFY93 Mat a ura3 leu2 lys2 vps27::KanMX
VPS28-GFP::HIS3
This study
SFY97
Mat α ura3 trp1 met15 pkh2::LEU2
pkh1-ts VPS28-GFP::HIS3
This study
SFY98 Mat a ura3 trp1 met15 pkh2::LEU2
VPS28-GFP::HIS3
This study
SFY99
Mat α ura3 leu2 VPS28-GFP::HIS3
This study
Pkh2-GFP
Mat a his31 leu20 met15 ura30
PKH2-GFP::HIS3
EUROSCARF
SFY133
Mat α his31 leu20 lys20
ura30 PKH2-GFP::HIS3
vps4::KanMX4
This study
SFY136
his31 leu20 lys20 ura30
PKH2-GFP::HIS3 vps27:: KanMX
vps4::KanMX4
This study
Vps27-GFP
Mat a his31 leu20 met15 ura30
VPS27-GFP::HIS3
EUROSCARF
SFY95 Mat a his3 leu2 ura3 VPS27-
GFP::HIS3 pkh2::LEU2 pkh1-ts
This study
SFY96 Mat a his3 leu2 ura3 met15 VPS27-
GFP::HIS3 pkh2::LEU2
This study
TABLE 1: Yeast strains.
Page 8
4062 | J. Morvan et al. Molecular Biology of the Cell
fragment containing the VPS27 gene amplified from genomic DNA
between NdeI and BamHI restriction sites of the pET15b plasmid.
The pSF55 plasmid bearing the K208R point mutation of Pkh2 was
obtained by site-directed mutagenesis of the pRH1250 plasmid us-
ing the GGTACGCCGCAAggGTACTAAAC and GTTTAGTACccT-
TGCGGCGTACC primers. pSF65, pSF66, and pSF69 plasmids
bearing the S613A mutation of Vps27 were obtained by site-
directed mutagenesis of pSF51, pDB17, and pDL56, respectively,
using the GAAAGGCCGCCTgcTCCTCAAGAGG and CCTCTT-
GAGGAgcAGGCGGCCTTTC primers, whereas pSF68, pSF70, and
pSF71 plasmids bearing the S613D mutation were obtained using
the GAAAGGCCGCCTgaTCCTCAAGAGG and CCTCTTGAGGAt-
cAGGCGGCCTTTC primers.
Protein extracts and Western blotting
Cells extracts were prepared by trichloroacetic acid precipitation
and NaOH lysis and proteins analyzed by immunoblotting as previ-
ously described (Volland et al., 1994).
Carboxypeptidase Y secretion assay
A 5-μl drop of culture at OD
600
of 0.4 was spotted on a YPD plate,
left to dry, and then covered with a water-hydrated nitrocellulose
membrane (Protran BA85; Whatman, Piscataway, NJ). After 48 h of
growth at 30°C the membrane was removed and rinsed with water,
and CPY secretion was revealed by immunoblotting with rabbit
polyclonal anti-CPY antibodies (a gift from H. Riezman, University of
Geneva, Switzerland).
6xHis-tagged protein purification
A 200-ml amount of LB/ampicillin/chloramphenicol was inoculated
with BL21 Rosetta (Novagen, Gibbstown, NJ) transformed with
pET15b-6His-VPS27. Cells were grown until OD
600
of 0.5 was
reached. Recombinant protein synthesis was induced by addition of
1 mM isopropyl-β-d-thiogalactoside for 2 h at 37°C. Cells were then
harvested, snap frozen, and thawed and then resuspended in 2 ml
of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM imidazole)
supplemented with protease inhibitor (Complete Mini-EDTA Free;
Roche, Indianapolis, IN). Cells were then lysed by sonication on ice
five times for 30 s with 30-s breaks. Insoluble material was removed
by centrifugation for 10 min at 13,000 × g. Soluble material was
loaded on a 500-μl bed volume Ni-Sepharose column (HisTrap HP;
GE Healthcare, Piscataway, NJ) equilibrated with lysis buffer. The
column was then washed twice with 500 μl of 50 mM imidazole buf-
fer (50 mM Tris, pH 7.5, 150 mM NaCl) and once with 500 μl of
80 mM imidazole buffer. The 6xHis-tagged proteins were eluted by
three times 500 μl of 500 mM imidazole buffer. Protein concentra-
tion was measured using Bradford reagent (Protein Assay; Bio-Rad,
Hercules, CA). The most concentrated fraction was dialyzed over-
night against 500 ml of 40 mM 3-(N-morpholino)propanesulfonic
acid (MOPS)/KOH, pH 7.4, 10 mM MgCl
2
, and 50% glycerol. Dia-
lyzed proteins were stored at –80°C.
In vitro phosphorylation assay
The pkh1 strain transformed with empty plasmid (YEp195),
YEp195-PKH2-3xHA, or YEp195-PKH2
K208R
-3xHA was grown until
exponential phase of growth at 30°C in SC selective medium. The
equivalent of 30 OD
600
units of cells was lysed in 500 μl of lysis
buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
pH7.5, 150 mM KCl, 1 mM EDTA, pH 7.5, 1 mM ethylene glycol
tetraacetic acid [EGTA], pH 7.5, 10% glycerol) supplemented
with protease inhibitor (Complete Mini-EDTA Free) with 500 μl of
glass beads by vortexing for 4 min. The lysate was cleared by two
acids, 2% glucose, and the appropriate CSM [MP Biomedicals,
Solon, OH] dropout mix).
The SFY93, SFY97, SFY98, SFY99, SFY102, SFY103, and SFY104
strains were obtained by crossing BY4741 VPS28-GFP (European
Saccharomyces cerevisiae Archive for Functional Analysis
[EUROSCARF], Institute for Molecular Biosciences, Johann Wolfgang
Goethe-University Frankfurt, Frankfurt, Germany) with INA106-3D.
The SFY93 strain was obtained by crossing the BY4742 vps27
strain with the BY4741 VPS28-GFP strain. The SFY133 strain was
obtained by crossing BY4742 vps4 (EUROSCARF) with BY4741
PKH2-GFP. The SFY95 and SFY96 strains were obtained by crossing
BY4741 VPS27-GFP (EUROSCARF) with INA106-3D.
The pSF51 and pSF52 plasmids were generated by cloning PCR
fragments containing respectively VPS27 endogenous promoter
and VPS27 gene full length or until nucleotide 1569, amplified
from pDL56, between the EcoRI and BamHI restriction sites of
YCpHAC33. The pDB17 plasmid was generated by cloning a PCR
Name Description Origin
pTS81 YEp195-PKH2-3xHA,
URA3, 2 μm
T. Schmelzle and M.
N. Hall, University of
Basel, Switzerland
pSF55 YEp195-pkh2
K208R
-3xHA,
URA3, 2 μm
This study
pDL56 pRS426-Prom-VPS27-
3xHA, URA3, 2 μm
R. C. Piper, University
of Iowa
pSF51 YCpHAC33-Prom-VPS27-
3xHA, URA3, CEN
This study
pSF52 YCpHAC33-Prom-
vps27
Cter
-3xHA, URA3,
CEN
This study
pSF65 YCpHAC33-Prom-vps-
27
S613A
-3xHA, URA3, CEN
This study
pSF68 YCpHAC33-Prom-
vps27
S613D
-3xHA, URA3,
CEN
This study
pSF69 pRS426- Prom-vps27
S613A
-
3xHA, URA3, 2 μm
This study
pSF70 pRS426- Prom-vps27
S613D
-
3xHA, URA3, 2 μm
This study
pDB17 pET15b-6xHis-VPS27,
Amp, Ori
This study
pSF66 pET15b-6xHis-vps27
S613A
,
Amp, Ori
This study
pSF71 pET15b-6xHis-vps27
S613D
,
Amp, Ori
This study
pGO45 pRS426-GFP-Cps1, URA3,
2 μm
Odorizzi et al. (1998)
pGO47 pRS424-GFP-Cps1, TRP1,
2 μm
Odorizzi et al. (1998)
pSte2-GFP pRS424-Ste2-GFP, TRP1,
2 μm
Stefan and Blumer
(1999)
pJMG282 pRS415-prom ADH-GFP-
VPS23, LEU2, CEN
Bugnicourt et al.
(2004)
TABLE 2: Plasmids.
Page 9
Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4063
centrifugations at 500 × g. The total lysate protein concentration
was quantified by Bradford reagent (Bio-Rad). Volume of total lysate
corresponding to 1 mg of proteins was brought up to 900 μl with
immunoprecipitation (IP) buffer (50 mM Tris/HCl, pH 7.5, 150 mM
NaCl, 1 mM EDTA, pH 7.5, 1 mM EGTA, pH 7.5, 1% Nonidet P-40)
supplemented with protease inhibitor (Complete Mini-EDTA Free),
and 50 μl of 50% protein G–Sepharose beads (Sigma-Aldrich, St.
Louis, MO) and 5 μl of rat anti-HA antibodies (3F10; Roche) were
added. The tubes were incubated on an overhead rotator overnight
at 4°C. The beads were washed three times with IP buffer and then
twice with 40 mM MOPS, pH 7.5.
The beads were then resuspended in 60 μl of phosphorylation
buffer (40 mM MOPS, pH 7.5, 10 mM MgCl
2
, 1 mM dithiothreitol).
A 10-μl amount of immune complex beads was mixed with 25 μg of
substrate protein in 10 μl of phosphorylation buffer and 2 μl of ATP
mix (1 mM ATP, 4 μCi of [γ-
32
P]ATP). The phosphorylation mixture
was incubated for 30 min at room temperature. The reaction was
stopped by addition of 4× Laemmli buffer containing 50 mM ATP.
The samples were boiled for 5 min and then loaded on a 10% poly-
acrylamide gel. The gel was treated for 5 min in 12.5% TCA and for
5 min in 50% (vol/vol) EtOH and 10% (vol/vol) acetic acid, dried, and
exposed on a phosphoscreen. After 24 h of exposure the screen was
scanned using a Typhoon Trio (GE Healthcare).
Subcellular fractionation
The equivalent of 30 OD
600
units of cells was lysed in 500 μl of
phosphate-buffered saline (PBS) and 0.25 M sorbitol supplemented
with protease inhibitor (Complete Mini-EDTA Free) with 500 μl of
glass beads by vortexing for 4 min at 4°C. The lysate was cleared by
two centrifugations of 3 min at 500 × g. The cleared lysate was then
spun for 10 min at 13,000 × g, generating the P13 pellet; the S13
was further spun for 1 h at 100,000 × g to generate the P100 and
S100. The P13 and P100 were resuspended in the same volume as
S100 of PBS, 0.25 M sorbitol, and 1% Triton X-100.
Equal volumes of each fraction were analyzed by SDS–PAGE,
followed by immunoblot with mouse monoclonal anti-Vps10 (Invit-
rogen, Carlsbad, CA), mouse monoclonal anti-HA (Roche), mouse
anti-ALP (Invitrogen), mouse anti-Vti1 (a gift from G. F. von Mollard,
Universität Bielefeld, Germany), and mouse anti-Pgk1 (Invitrogen)
antibodies.
Fluorescence microscopy
Cells expressing the different GFP-tagged proteins were grown to
mid-exponential growth phase in selective medium before obser-
vation in the selective medium using fluorescence microscopy
(Axiovert 200, 100× objective, differential interference contrast
and GFP filters [Carl Zeiss, Jena, Germany]). Images were acquired
with AxioVision (Zeiss) software using a CoolSnapHQ2 camera
(Roper Scientific, Tucson, AZ) and processed with ImageJ software
(National Institutes of Health, Bethesda, MD).
ACKNOWLEDGMENTS
We thank Naima Belgareh-Touzé, Scott D. Emr, Rosine Haguenauer-
Tsapis, Robert C. Piper, Howard Riezman, and Gabriele Fischer von
Mollard for sharing antibodies, strains, and plasmids; J. O. De
Craene and N. Joly for critical reading of the manuscript; the Friant
laboratory’s members for support; and Romeo Ricci for support dur-
ing the revision process. This work was supported by the Centre
National de la Recherche Scientifique (ATIP-CNRS 05-00932 and
ATIP-Plus 2008-3098 to S.F.), the Agence Nationale de la Recherche
(ANR-07-BLAN-0065 to S.F.), the Fondation Recherche Médicale
(FRM INE20051105238 and FRM-Comité Alsace 2006CX67-1 to
REFERENCES
Albuquerque CP, Smolka MB, Payne SH, Bafna V, Eng J, Zhou H (2008). A
multidimensional chromatography technology for in-depth phosphopro-
teome analysis. Mol Cell Proteomics 7, 1389–1396.
Bilodeau PS, Winistorfer SC, Kearney WR, Robertson AD, Piper RC (2003).
Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of
sorting ubiquitinated proteins at the endosome. J Cell Biol 163, 237–243.
Boura E, Rozycki B, Herrick DZ, Chung HS, Vecer J, Eaton WA, Cafiso DS,
Hummer G, Hurley JH (2011). Solution structure of the ESCRT-I complex
by small-angle x-ray scattering, EPR, and FRET spectroscopy. Proc Natl
Acad Sci USA 108, 9437–9442.
Bugnicourt A, Froissard M, Sereti K, Ulrich HD, Haguenauer-Tsapis R,
Galan JM (2004). Antagonistic roles of ESCRT and Vps class C/HOPS
complexes in the recycling of yeast membrane proteins. Mol Biol Cell
15, 4203–4214.
Casamayor A, Torrance PD, Kobayashi T, Thorner J, Alessi DR (1999).
Functional counterparts of mammalian protein kinases PDK1 and SGK in
budding yeast. Curr Biol 9, 186–197.
Chen P, Lee KS, Levin DE (1993). A pair of putative protein kinase genes
(YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae.
Mol Gen Genet 236, 443–447.
deHart AK, Schnell JD, Allen DA, Hicke L (2002). The conserved Pkh-Ypk
kinase cascade is required for endocytosis in yeast. J Cell Biol 156,
241–248.
Feng Y, Davis NG (2000). Akr1p and the type I casein kinases act prior to
the ubiquitination step of yeast endocytosis: Akr1p is required for kinase
localization to the plasma membrane. Mol Cell Biol 20, 5350–5359.
Friant S, Lombardi R, Schmelzle T, Hall MN, Riezman H (2001). Sphingoid
base signaling via Pkh kinases is required for endocytosis in yeast.
EMBO J 20, 6783–6792.
Friant S, Zanolari B, Riezman H (2000). Increased protein kinase or de-
creased PP2A activity bypasses sphingoid base requirement in endocy-
tosis. EMBO J 19, 2834–2844.
Gietz D, St Jean A, Woods RA, Schiestl RH (1992). Improved method for
high efficiency transformation of intact yeast cells. Nucleic Acids Res 20,
1425.
Gill DJ, Teo H, Sun J, Perisic O, Veprintsev DB, Emr SD, Williams RL (2007).
Structural insight into the ESCRT-I/-II link and its role in MVB trafficking.
EMBO J 26, 600–612.
Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faergeman NJ, Mann
M, Jensen ON (2005). Quantitative phosphoproteomics applied to the
yeast pheromone signaling pathway. Mol Cell Proteomics 4, 310–327.
Hanafusa H, Ishikawa K, Kedashiro S, Saigo T, Iemura S, Natsume T,
Komada M, Shibuya H, Nara A, Matsumoto K (2011). Leucine-rich
repeat kinase LRRK1 regulates endosomal trafficking of the EGF
receptor. Nat Commun 2, 158.
Hicke L, Zanolari B, Riezman H (1998). Cytoplasmic tail phosphorylation of
the alpha-factor receptor is required for its ubiquitination and internal-
ization. J Cell Biol 141, 349–358.
Hierro A, Sun J, Rusnak AS, Kim J, Prag G, Emr SD, Hurley JH (2004).
Structure of the ESCRT-II endosomal trafficking complex. Nature 431,
221–225.
Hoeller D et al. (2006). Regulation of ubiquitin-binding proteins by monou-
biquitination. Nat Cell Biol 8, 163–169.
Hurley JH (2008). ESCRT complexes and the biogenesis of multivesicular
bodies. Curr Opin Cell Biol 20, 4–11.
Im YJ, Wollert T, Boura E, Hurley JH (2009). Structure and function of the
ESCRT-II-III interface in multivesicular body biogenesis. Dev Cell 17,
234–243.
Inagaki M, Schmelzle T, Yamaguchi K, Irie K, Hall MN, Matsumoto K (1999).
PDK1 homologs activate the Pkc1-mitogen-activated protein kinase
pathway in yeast. Mol Cell Biol 19, 8344–8352.
Katzmann DJ, Stefan CJ, Babst M, Emr SD (2003). Vps27 recruits ESCRT
machinery to endosomes during MVB sorting. J Cell Biol 162, 413–423.
Lata S, Schoehn G, Jain A, Pires R, Piehler J, Gottlinger HG, Weissenhorn W
(2008). Helical structures of ESCRT-III are disassembled by VPS4. Science
321, 1354–1357.
Lauwers E, Erpapazoglou Z, Haguenauer-Tsapis R, Andre B (2010). The ubiq-
uitin code of yeast permease trafficking. Trends Cell Biol 20, 196–204.
Marchal C, Dupre S, Urban-Grimal D (2001). Casein kinase I controls a late
step in the endocytic trafficking of yeast uracil permease. J Cell Sci 115,
217–226.
S.F.), and the Association pour la Recherche sur le Cancer (ARC JR/
MLD/MDV-CR306/7901 to S.F.).
Page 10
4064 | J. Morvan et al. Molecular Biology of the Cell
Smolka MB, Albuquerque CP, Chen SH, Zhou H (2007). Proteome-wide
identification of in vivo targets of DNA damage checkpoint kinases. Proc
Natl Acad Sci USA 104, 10364–10369.
Steen H, Kuster B, Fernandez M, Pandey A, Mann M (2002). Tyrosine phos-
phorylation mapping of the epidermal growth factor receptor signaling
pathway. J Biol Chem 277, 1031–1039.
Stefan CJ, Blumer KJ (1999). A syntaxin homolog encoded by VAM3
mediates down-regulation of a yeast G protein-coupled receptor. J Biol
Chem 274, 1835–1841.
Stern KA, Visser Smit GD, Place TL, Winistorfer S, Piper RC, Lill NL (2007).
Epidermal growth factor receptor fate is controlled by Hrs tyrosine
phosphorylation sites that regulate Hrs degradation. Mol Cell Biol 27,
888–898.
Teo H, Gill DJ, Sun J, Perisic O, Veprintsev DB, Vallis Y, Emr SD, Wil-
liams RL (2006). ESCRT-I core and ESCRT-II GLUE domain structures
reveal role for GLUE in linking to ESCRT-I and membranes. Cell 125,
99–111.
Urbe S, Mills IG, Stenmark H, Kitamura N, Clague MJ (2000). Endosomal
localization and receptor dynamics determine tyrosine phosphorylation
of hepatocyte growth factor-regulated tyrosine kinase substrate. Mol
Cell Biol 20, 7685–7692.
Volland C, Urban-Grimal D, Geraud G, Haguenauer-Tsapis R (1994). Endo-
cytosis and degradation of the yeast uracil permease under adverse
conditions. J Biol Chem 269, 9833–9841.
Williams RL, Urbe S (2007). The emerging shape of the ESCRT machinery.
Nat Rev Mol Cell Biol 8, 355–368.
Wollert T, Yang D, Ren X, Lee HH, Im YJ, Hurley JH (2009). The ESCRT
machinery at a glance. J Cell Sci 122, 2163–2166.
Zanolari B, Friant S, Funato K, Sutterlin C, Stevenson BJ, Riezman H (2000).
Sphingoid base synthesis requirement for endocytosis in Saccharomyces
cerevisiae. EMBO J 19, 2824–2833.
Marchal C, Haguenauer-Tsapis R, Urban-Grimal D (1998). A PEST-like
sequence mediates phosphorylation and efficient ubiquitination of yeast
uracil permease. Mol Cell Biol 18, 314–321.
Marchal C, Haguenauer-Tsapis R, Urban-Grimal D (2000). Casein kinase
I-dependent phosphorylation within a PEST sequence and ubiquitination
at nearby lysines signal endocytosis of yeast uracil permease. J Biol Chem
275, 23608–23614.
Obita T, Saksena S, Ghazi-Tabatabai S, Gill DJ, Perisic O, Emr SD, Williams
RL (2007). Structural basis for selective recognition of ESCRT-III by the
AAA ATPase Vps4. Nature 449, 735–739.
Odorizzi G, Babst M, Emr SD (1998). Fab1p PtdIns(3)P 5-kinase func-
tion essential for protein sorting in the multivesicular body. Cell 95,
847–858.
Pornillos O, Alam SL, Davis DR, Sundquist WI (2002). Structure of the
Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6
protein. Nat Struct Biol 9, 812–817.
Reggiori F, Pelham HR (2001). Sorting of proteins into multivesicular bod-
ies: ubiquitin-dependent and -independent targeting. EMBO J 20,
5176–5186.
Ren X, Hurley JH (2011). Structural basis for endosomal recruitment of
ESCRT-I by ESCRT-0 in yeast. EMBO J 30, 2130–2139.
Ren X, Kloer DP, Kim YC, Ghirlando R, Saidi LF, Hummer G, Hurley JH
(2009). Hybrid structural model of the complete human ESCRT-0 com-
plex. Structure 17, 406–416.
Robinson LC, Menold MM, Garrett S, Culbertson MR (1993). Casein kinase
I-like protein kinases encoded by YCK1 and YCK2 are required for yeast
morphogenesis. Mol Cell Biol 13, 2870–2881.
Roth AF, Papanayotou I, Davis NG (2011). The yeast kinase Yck2 has a
tripartite palmitoylation signal. Mol Biol Cell 22, 2702–2715.
Saksena S, Sun J, Chu T, Emr SD (2007). ESCRTing proteins in the endocytic
pathway. Trends Biochem Sci 32, 561–573.
Page 11
  • Source
    • "proteasome-mediated degradation of ESCRT-0 is not comparable to a deletion of VPS27, since there is no initial substrate recognition by ESCRT-0 in this case. We also note that despite work showing that Vps27 is phosphorylated by the Pkh1/2 kinases to control Cps1 and PM protein trafficking to the MVB (Morvan et al., 2012 ), we did not identify these kinases in our genetic screen (Table S1 ), nor did phosphomimic or non-phosphorylated forms of Vps27 alter vacuolar sorting of GFP-Yif1 upon starvation (data not shown). Thus, Vps27 may undergo differential regulation by multiple signals that could potentially modify its ability to recognize substrates under different conditions of growth. "
    [Show abstract] [Hide abstract] ABSTRACT: Upon amino acid (AA) starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB) pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC) process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC) complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.
    Full-text · Article · Sep 2015 · Cell Reports
  • Source
    • "The ubiquitinated Ste2 proteins are then delivered to the late endosome/multivesicular body (MVB) compartment in which they are sorted for delivery to the intralumenal vesicles (ILV) for final degradation in the vacuole (Stefan and Blumer 1999 ). Based on the result that Ste2-cargo sorting on endosomal membrane require the activity of ESCRT (endosomal sorting complex required for transport) (Morvan et al. 2012), we reasoned that the slow Ste2-GFP traffic toward the vacuole in Vps1-deficient cells was probably due to a disruption of ESCRT integrity. Our approach to define the functional relationship between Vps1 and subunits of ESCRT was producing combination mutants in two genes to observe genetic interactions. "
    [Show abstract] [Hide abstract] ABSTRACT: Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 delta cells accumulated FM4-64 to a greater extent than wild-type (WT))cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.
    Full-text · Article · Mar 2013 · Journal of Biosciences
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain containing proteins involved in protein trafficking between the Golgi and late endosomes (LE). They interact with clathrin, clathrin adaptor at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3/Tda3, a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten disease linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the LE to the Golgi. We show that Btn3 endosomal localization depends on epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitinated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten disease linked protein Btn2.
    Full-text · Article · Feb 2015 · Journal of Cell Science
Show more