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Developmental Cell
Article
The CORVET Tethering Complex Interactswith the Yeast Rab5 Homolog Vps21and Is Involved in Endo-Lysosomal BiogenesisKarolina Peplowska,1 Daniel F. Markgraf,1,3 Clemens W. Ostrowicz,1,3 Gert Bange,2 and Christian Ungermann1,*1 University of Osnabruck, Department of Biology, Biochemistry Section, Barbarastrasse 13, 49076 Osnabruck, Germany2 Biochemie Zentrum der Universitat Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany3 These authors contributed equally to this work.*Correspondence: [email protected]
DOI 10.1016/j.devcel.2007.03.006
SUMMARY
The dynamic equilibrium between vesicle fis-sion and fusion at Golgi, endosome, and vacu-ole/lysosome is critical for the maintenance oforganelle identity. It depends, among others,on Rab GTPases and tethering factors, whosefunction and regulation are still unclear. Wenow show that transport among Golgi, endo-some, and vacuole is controlled by two homol-ogous tethering complexes, the previouslyidentified HOPS complex at the vacuole anda novel endosomal tethering (CORVET) com-plex, which interacts with the Rab GTPaseVps21. Both complexes share the four classC Vps proteins: Vps11, Vps16, Vps18, andVps33. The HOPS complex, in addition, con-tains Vps41/Vam2 and Vam6, whereas theCORVET complex has the Vps41 homologVps8 and the (h)Vam6 homolog Vps3. Strikingly,the CORVET and HOPS complexe can intercon-vert; we identify two additional intermediatecomplexes, both consisting of the class Ccore bound to Vam6-Vps8 or Vps3-Vps41. Ourdata suggest that modular assembled tetheringcomplexes define organelle biogenesis in theendocytic pathway.
INTRODUCTION
Vesicle-mediated protein transport between organelles of
the endomembrane system depends on a dynamic equi-
librium between fission and fusion. Alterations of this bal-
ance lead to a loss of organelle identity and subsequently
to disease (Di Pietro and Dell’Angelica, 2005; Munro,
2004). Vesicle fusion requires Rab GTPase-dependent
tethering of the vesicle, followed by the fusion process,
which is driven by SNARE proteins residing on the vesicle
and organelle membrane. Rab GTPases have been impli-
cated as key regulators of fusion (Grosshans et al., 2006).
To be able to bind their effectors, Rab GTPases need to be
Deve
converted from the inactive GDP to the active GTP form.
The conversion depends on guanine nucleotide exchange
factors (GEFs), which vary in size and domain compo-
sition. Inactivation of Rab proteins depends on Rab
GTPase-activating proteins (GAPs), which trigger GTP
hydrolysis (Haas et al., 2005).
Tethering factors or large multimeric tethering com-
plexes (tethers) cooperate with Rab GTPases to capture
vesicles and trap them prior to the action of SNAREs (Beh-
nia and Munro, 2005; Grosshans et al., 2006). Tethers are
therefore thought to coordinate Rab and SNARE function
and provide an essential layer of specificity to fusion reac-
tions. Several large tethering complexes have been iden-
tified, including the exocyst at the plasma membrane,
the TRAPP and the COG complex at the Golgi, the
GARP complex at endosomes, and the HOPS complex
at the vacuole (Grosshans et al., 2006; Whyte and Munro,
2002). Interestingly, some tethers have been identified as
Rab GEFs, including the TRAPP complex and the Vam6
subunit of the HOPS complex (Wang et al., 2000; Wurmser
et al., 2000). A number of tethering complexes, which bind
to specific Rab GTPases, have been characterized to
date. However, since many of them harbor numerous
subunits and multiple domains, their precise function
remains widely elusive.
We are interested in tethering within the endolysosomal
system. In yeast (and later in mammalian cells), the multi-
subunit HOPS/class C Vps complex was identified as
a tethering complex, which is required for homotypic fu-
sion at the vacuole. It consists of six proteins: Vps41
(Vam2) and Vam6 (Vps39) and the class C subunits
Vps11, Vps16, Vps18, and Vps33 (Nakamura et al.,
1997; Price et al., 2000b; Rieder and Emr, 1997; Seals
et al., 2000; Wurmser et al., 2000). The complex interacts
with the GTP form of Ypt7 (yeast Rab7) and can bind to
SNAREs (Collins et al., 2005; Laage and Ungermann,
2001; Stroupe et al., 2006). It is therefore thought that
HOPS mediates the transition from tethering to trans-
SNARE pairing during fusion. The individual subunits
have strikingly different domains. Vps33 is homologous
to Sec1/Munc18 proteins, Vps11 and Vps18 have essen-
tial RING domains at their C termini (Rieder and Emr,
1997), and Vps16 has two conserved domains of unknown
function (Richardson et al., 2004) (http://www.pfam.org).
lopmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 739
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 1. Characterization of the vps3D and vps8D Mutants
(A) Localization of vacuolar protein markers in vps3D cells. Wt and vps3D cells expressing GFP-tagged vacuolar proteins were grown logarithmically
in YPD medium, harvested, washed once with PBS buffer, and analyzed by fluorescence microscopy. Size bar = 5 mm.
(B) Double mutant of vps8 and vam3. Cells lacking Vps8, Vam3, or both were stained with FM4-64 and analyzed by fluorescence microscopy. Size
bar = 10 mm.
740 Developmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc.
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Vps41 (Vam2) seems to have two roles, one in tethering
(as part of the HOPS complex) (Price et al., 2000a; Price
et al., 2000b) and the other in the biogenesis of AP-3 ves-
icles, which transport selected vacuolar proteins from the
Golgi to the vacuole (Rehling et al., 1999). Finally, the
HOPS subunit Vam6 acts as a GEF on the Rab GTPase
Ypt7 (Wurmser et al., 2000). Overexpression of the human
Vam6 leads to clustering of lysosomes, suggesting a direct
role in tethering (Caplan et al., 2001). Interestingly, mam-
malian cells contain two homologs of Vam6, hVps39/
TLP (Caplan et al., 2001; Felici et al., 2003) and TRAP-1
(Charng et al., 1998). Both are expressed in all tissues
and have been implicated in selective Smad signaling at
the endosome.
Deletion of any class C gene leads to severe fragmenta-
tion of the vacuole (Raymond et al., 1992; Rieder and Emr,
1997). In fact, the class C proteins function not only at the
vacuole, but also at endosomes (Peterson and Emr, 2001;
Srivastava et al., 2000), where they have been shown to
bind Vps8 instead of Vps41 and Vam6 (Subramanian
et al., 2004). Vps8 is conserved across species and is crit-
ical for sorting of proteins to the endosome but dispens-
able for the AP-3 pathway (Chen and Stevens, 1996;
Horazdovsky et al., 1996). It belongs to the class D VPS
genes, whose deletion leads to an enlarged vacuole phe-
notype. Intriguingly, a number of proteins implicated in
vesicle fusion at the endosome, like Vps21 (yeast Rab5),
Vps9 (yeast Rabex5), the SNARE Pep12, Vac1 (homolo-
gous to EEA1), and Vps45 (Sec1-like protein), also belong
to this group (Bowers and Stevens, 2005). However, little
is known about Vps3, another class D gene. It was identi-
fied by Stevens and coworkers and implicated in Golgi
and endosome vesicle trafficking (Raymond et al., 1990).
We now show that Vps3 is homologous to (h)Vam6 and,
together with Vps8, is part of a novel tethering complex at
the endosome, which we term class C core vacuole/endo-
some tethering (CORVET) complex. We present evidence
that the CORVET and the HOPS complex can interconvert
by dynamic subunit exchange. Our data indicate a link
between modular assembled tethering complexes, Rab
GTPases, and the transition between endosomes and
lysosomes.
RESULTS
Organelle Identity of the Vacuole in Class D Mutants
We previously identified Vps3 as one of the proteins im-
paired in salt-induced vacuole fragmentation (Figure S1A;
LaGrassa and Ungermann, 2005). VPS3 belongs to the
class D genes, whose deletion results in a characteristic
phenotype of enlarged vacuoles (Figure S1B; Raymond
et al., 1990; Rothman et al., 1989). We wondered if defects
in vacuole fragmentation in class D mutants could be due
to the lack of vacuolar markers and loss of vacuolar
Deve
identity. Indeed, vacuoles in vps3D cells do not acidify
(Preston et al., 1989; Raymond et al., 1990; Rothman
et al., 1989) but show efficient localization of Yck3,
Vps41, Vam3, Vac8, or Pho8 (Figure 1A), suggesting that
the class D organelle has lysosome-like properties.
If the class D organelles resemble wild-type vacuoles,
they should require the vacuolar Q-SNARE Vam3 for in-
tegrity. However, we found that large class D organelles
of a vps8 mutant do not fragment, even if the vacuolar
SNARE VAM3 has been deleted (Figure 1B). This pheno-
type contrasts to the complete fragmentation of vacuoles
in the single vam3 deletion mutant (Nichols et al., 1997). It
is possible that the endosomal Q-SNARE Pep12 is taking
over the Vam3 function in the vam3D vps8D mutant (Gotte
and Gallwitz, 1997). In agreement with this observation,
we observed that the endosomal Rab5 homolog Vps21
and the vacuolar Rab Ypt7 colocalize in the vps8D mutant
(Figure 1C). Thus, class D vacuoles in vps8D or vps3D
mutants behave like endosome-vacuole hybrids, which
do not acidify, and have a deficiency in vacuole fragmen-
tation (Figure S1B) and vacuole inheritance (Figure 1D).
Vps3 Is Part of a Novel Endosomal
Tethering Complex
To understand the biogenesis of this putative hybrid or-
ganelle on the molecular level, we addressed the localiza-
tion and function of Vps3, the least characterized of all
class D genes. By subcellular localization, Vps3 was re-
covered in equal amounts in membrane and soluble frac-
tions, and its steady-state localization was not influenced
by the deletion of Vam6, Vps8, or Vps21 (data not shown).
In agreement with its proposed endosomal function, we
found GFP-tagged Vps3 in dot-like structures (Figure S2).
To identify potential interaction partners, we chromoso-
mally tagged Vps3 at the C terminus with the tandem affin-
ity purification (TAP) tag and isolated the protein from
yeast cells on IgG Sepharose and calmodulin (CaM) beads
(Figure 2A). Several bands were specifically retained with
Vps3, and their identity was determined by mass spec-
trometry. Surprisingly, we found all four class C proteins
(Vps33, Vps16, Vps11, Vps18) and Vps8. The association
of Vps8 with some class C proteins has been previously
identified and implicated in Golgi-to-endosome transport
(Subramanian et al., 2004). We therefore asked whether
Vps3 associates with Vps8 and the class C proteins indi-
vidually or forms a complex with these proteins. To distin-
guish between these possibilities, we generated yeast de-
tergent extracts to determine the size of Vps3 and Vps8.
Using glycerol gradient centrifugation, we detected Vps3
and Vps8 at a molecular weight of 700 and 120 kDa
(data not shown), supporting the idea of both proteins be-
ing part of the 700 kDa high molecular weight complex. To
rule out the possibility that the two proteins comigrate in
the glycerol gradient due to homo-oligomerization, we
(C) Colocalization of Vps21 and Ypt7. RFP-Ypt7 and GFP-Vps21 were expressed in wt and vps8D cells and visualized by fluorescence microscopy.
Images were processed by deconvolution using the Autoquant software. Size bar = 10 mm.
(D) Vacuole inheritance. Wt and vps3D cells were logarithmically grown in YPD medium, stained with FM4-64 for 30 min, reisolated, and grown in fresh
YPD medium for 3 hr before being analyzed by fluorescence microscopy. Quantification of at least 200 cells of each strain is shown.
lopmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 741
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 2. Identification of the Vps3-
Vps8-Class C Complex
(A) Vps3-TAP purification. BJ3505 cells (wt and
VPS3-TAP) were lysed and the Vps3-TAP pro-
tein was purified according to the TAP protocol
(see Experimental Procedures). The eluate was
loaded onto a 4%–12% SDS-PAGE gradient
gel and candidate proteins were identified by
mass spectroscopy. Vps3-cbp (CaM-binding
peptide) is left after TEV cleavage.
(B–D) Sizing of Vps3-8-class C complex by gel
filtration (B). BY4741 cells carrying Vps3-TAP
were lysed and Vps3-TAP was captured on
IgG-Sepharose. After TEV cleavage, the eluate
was applied onto a Superose 6 column, and
proteins in fractions 6–19 were analyzed on
a 4%–12% SDS-PAGE gradient gel, followed
by Coomassie staining. Bands were identified
by mass spectrometry. The same purification
from BY4741 VPS8-TAP cells is shown in (C).
As a comparison, Vps41-TAP was purified by
the same approach (D). Models of the com-
plexes are shown. Class C = Vps11, Vps16,
Vps18, Vps33; 8 = Vps8; 3 = Vps3; 41 =
Vps41; V6 = Vam6.
decided to repeat the TAP purification and determine the
mobility of the Vps3 and the Vps8 complex using gel filtra-
tion. As shown in Figure 2B, Vps3 is found in a 700 kDa
complex together with Vps8, Vps11, Vps16, Vps18, and
Vps33. We also observe Vps3 in a second peak at around
120 kDa. To determine if Vps3 dissociated partially from
the Vps8-class C complex during the chromatography
step, we used Vps8 as the bait in our purification
(Figure 2C). Strikingly, the purification yielded a very sim-
ilar picture, with Vps3 being present in the Vps8-class C
complex and as a putative monomer. Vps3 must have
been in a complex with Vps8 initially to be present on
the gel filtration column and most likely dissociated par-
tially from the complex during the column run, probably
due to its distinct binding characteristics to the other sub-
units. This observation is underscored by the equimolar
amounts of Vps3 and the other subunits. We therefore
conclude that Vps3 is a subunit of a novel endosomal
complex. Given that the complex contains the class C
tethering proteins, functions at the endosome, and is re-
quired for transport between endosome and vacuole
(see below), we term it CORVET complex (class C core
vacuole/endosome tethering).
742 Developmental Cell 12, 739–750, May 2007 ª2007 Elsevie
CORVET Interacts with Vps21 (Yeast Rab5)
The CORVET complex exhibits a remarkable similarity to
the HOPS complex (depicted in the models in Figures
2B and 2D). Both complexes are composed of six sub-
units, four of which—the class C proteins Vps11, Vps16,
Vps18, and Vps33—are found in both. Whereas the
HOPS complex also contains Vps41 and Vam6, the novel
complex has Vps8 and Vps3 instead. Both complexes
have the same size (Figure 2D). Moreover, Vps8 function-
ally interacts with the endosomal Rab5 homolog Vps21
(Horazdovsky et al., 1996). Since the HOPS complex has
been shown to be an effector of the Rab Ypt7 (Seals
et al., 2000; unpublished data), we analyzed the interac-
tion of the CORVET complex with Vps21. As shown in
Figure 3A, purified CORVET binds exclusively to Vps21-
GTP by pull-down.
Intriguingly, Vps3 shows homology to the human and
yeast Vam6 protein but contains an additional N-terminal
domain, which is not found in any other Vam6 homolog
(Figure S3). When this domain was excluded from the
alignment, we could observe a clear homology to hVam6
(Figure S3). Since Vam6 interacts with Ypt7-GDP and
can promote nucleotide exchange (Wurmser et al.,
r Inc.
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 3. Interaction of the Rab-GTPase
Vps21 with CORVET and Vps3
(A) Interaction of CORVET with Vps21. COR-
VET was purified from a strain overexpressing
Vps3, similar to Figure 2B, and the complex
fraction (11, 12) was applied to immobilized
GST-Vps21, Ypt1, and Ypt7, which were pre-
loaded with the indicated nucleotide (NF, nu-
cleotide-free). Bound protein was eluted by
EDTA/high salt, TCA precipitated, and ana-
lyzed by SDS-PAGE and western blotting using
Protein A-peroxidase. The Rab-GTPase was
eluted by boiling beads in sample buffer and
analyzed as above. Note that purified CORVET
complex lacks most Vps3 due to its dissocia-
tion during chromatography (Figure 2B).
(B and C) Interaction of Vps3 with Vps21. De-
tergent lysate prepared from 3 l of cells overex-
pressing Vps3 in the presence (B) or absence
(C) of Vps8 was applied to GST-Rabs contain-
ing the indicated nucleotide. Analysis was
done as in (A).
(D and E) Localization of GFP-Vps21. Cells
lacking or overexpressing Vps8, Vps3, or
Vam6 in the presence of GFP-Vps21 were
analyzed by fluorescence microscopy as in
Figure 1A. Size bar = 10 mm.
2000), we asked whether Vps3 can bind to Vps21-GDP.
When lysates from cells overexpressing Vps3 were ap-
plied to GST-Ypt7, Ypt1, or Vps21, Vps3 was recovered
with Vps21 but not Ypt7 or Ypt1 (Figure 3B). Surprisingly,
we did not observe a nucleotide-specific binding, sug-
gesting that we analyzed different Vps3 populations in
this assay (as a monomer and as part of the CORVET
complex). We therefore repeated the assay using lysates
from vps8D cells, which lack the CORVET complex,
and detected Vps3 preferentially in complex with the
nucleotide-free and GDP form of Vps21 (Figure 3C).
Likewise, Vps3 bound preferentially to the GDP/nucleo-
tide-free-mimicking S21N mutant of Vps21 in a co-
overexpression experiment (Figure S4). Thus, the Vps3
binds to Vps21-GDP, which mirrors the Vam6 binding to
Ypt7-GDP.
To test for a functional relationship of CORVET and
Vps21 in vivo, we followed GFP-Vps21 localization. Vps8
overexpression led to a striking dot-like accumulation of
Vps21 on membranes, whereas it appeared dispersed in
wild-type cells (Figures 3D and S5). This membrane accu-
mulation of GFP-Vps21 required Vps3 (Figure 3E), indicat-
ing that the CORVET subunits Vps8 and Vps3 cooperate in
Vps21 localization. Overexpression of Vps3 seemed to
have a similar effect, although the fragmentation of the
vacuoles (see below) interfered with a quantitative analy-
sis. In sum, the CORVET complex acts as an effector of
the Rab-Vps21, while the Vps3 protein has preference
for Vps21 in the nucleotide-free and GDP forms, which
is consistent with a GEF-like function.
Deve
Identification of Intermediate Tethering Complexes
It is striking that the CORVET and HOPS complex both
contain the class C core and have homologous subunits.
We therefore wondered whether intermediate complexes
might exist that could form during endosome-vacuole bio-
genesis. If Vps3 and Vam6 would exchange, two new
complexes with the class C core would be possible: one
containing Vam6-Vps8 and the other containing Vps3-
Vps41. To probe for their existence in wild-type cells, we
TAP-tagged Vam6 and Vps41 and looked for their associ-
ation with Vps3 and Vps8. Indeed, the Vam6-Vps8 com-
plex is present in wild-type cells (Figures 4A and 4B);
HA-tagged Vps8 copurified with Vam6-TAP and the class
C core on a gel filtration column. This association is not
apparent by Coomassie staining, since the HOPS com-
plex is the primary complex purified. Likewise, the Vps3-
Vps41 complex was detected in wild-type cells using
Vps41-TAP as bait, but not in vps33D cells (Figure 4C,
lane 1 versus 5; see below). Thus, the class C core is
a module found in four different complexes: (1) the
CORVET (Vps3-Vps8) complex, (2) together with Vam6
and Vps8 (i-HOPS, see below), (3) in the HOPS complex,
and (4) in combination with Vps3 and Vps41 (i-CORVET,
see below).
Dynamics of Tethering Complexes
Whereas the CORVET and HOPS complex appear to be
more abundant in the cell, the intermediate complexes
seem to be transient. We wondered whether we could
accumulate the intermediate complexes under certain
lopmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 743
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 4. Identification of Two Interme-
diate Complexes
(A and B) Identification of the Vam6-Vps8-class
C intermediate. Cells containing TAP-tagged
Vam6 and HA-tagged Vps8 were processed
for HOPS purification as in Figure 2B. Proteins
in each fraction were resolved on gradient
SDS-PAGE gels, which were either stained
with Coomassie ([A], 80% of the sample) or
transferred to nitrocellulose ([B], 20%). West-
ern blots were first decorated with antibodies
to the HA-tag and a mouse secondary anti-
body coupled to AlexaFluor680 (Molecular
Probes; [B], bottom panel), then with anti-
bodies against Vps41 coupled to a goat anti-
rabbit secondary antibody coupled to IR-
Dye800 (LI-COR). To visualize both proteins,
the channels were merged (top panel). A model
of the Vps8-Vam6-class C (i-HOPS) complex is
shown.
(C and D) Identification and dynamics of Vps3-
Vps41 complexes. Wt or deletion strains con-
taining Vps41-TAP (C) or Vps3-TAP (D) were
lysed. Tagged proteins were purified on CaM
beads using 700 mg of total protein (see Exper-
imental Procedures). EGTA eluates were TCA
precipitated, and proteins were analyzed on
a 7.5% SDS-PAGE gel, followed by western
blotting with antibodies against the tag, Vps3
or Vps41, Vps33, and Vac8. Note that both
the Vps33 and the Vps41 antibodies cross-re-
act in the total lysate with unknown proteins at
their apparent molecular weight. The proteins
are, however, not detected upon complex pu-
rification in the vps33D or vps41D background,
confirming that they are not Vps33 and Vps41,
respectively.
(E) Purification and sizing of the Vps3-Vps41-class C complex. TAP-tagged Vps3 was purified from vps8 cells as in Figure 2B. The proteins were an-
alyzed as in (A). The identity of Vps41 was confirmed by western blotting and mass spectrometry (data not shown). The model depicts the Vps3-
Vps41-class C (i-CORVET) complex.
conditions. To this end, we concentrated on the Vps3-
Vps41-class C intermediate (i-CORVET) and followed
the complex in several mutant strains lacking endosomal
or vacuolar proteins. Vps41 was TAP-tagged in wild-
type cells and the vps3, vam6, vps33, and vps8 deletion
mutants, and complexes were purified using CaM beads
(Figure 4C). In wild-type cells, the class C core subunit
Vps33 is associated with Vps41 (lane 1). As discussed,
a small amount of Vps3 was also detected, which was ab-
sent in a vps3D mutant (lane 5). This picture changed dra-
matically when Vam6 was absent: the association of Vps3
with Vps41 increased several-fold (lane 4). A similar asso-
ciation of Vps3 with Vps41 was also observed in the ab-
sence of Vps8 (lane 3). Vac8, a vacuolar fusion factor,
was not recovered in any complexes. Similar results
were obtained when Vps3 was tagged in mutant strains;
the level of the Vps3-Vps41 association was increased
several-fold in the vps8 or the vam6 mutant (Figure 4D).
To demonstrate the presence of an intermediate complex
containing Vps3, Vps41, and the class C proteins, we pu-
rified Vps3-TAP from the vps8D mutant. As shown in
Figure 4E, the complex is comparable to the HOPS and
CORVET complex in size and abundance, indicative of
744 Developmental Cell 12, 739–750, May 2007 ª2007 Elsev
an intermediate endosome-vacuole tethering complex.
Besides the identification of the Vps41-Vps3-class C in-
termediate complex, our data also indicate that Vps41
can replace Vps8. It is therefore likely that the proteins
may perform similar functions in the complexes.
Endosomal Vps3 Can Affect HOPS Function
and Vacuole Morphology
To rule out the possibility that Vps3 replaces Vam6 only if
Vam6 is absent from cells and to test if Vps3 can also com-
pete with Vam6 for Vps41 in wild-type cells, we overex-
pressed Vps3 and followed vacuole morphology and the
composition of the complex associated with Vps41. While
deletion of vps3 leads to a single enlarged vacuole in the
cell, overexpression of Vps3 caused complete vacuole
fragmentation (Figure 5A). This phenotype is strikingly
similar to the morphology of vam6D vacuoles (Wada
et al., 1992). We used the Vps41 protein as bait to analyze
the composition of the HOPS complex under these condi-
tions. In wild-type cells or when Vps3 expression was re-
pressed, Vam6 was associated with Vps41 (Figure 5B,
lane 1). However, when Vps3 was overproduced (lane 3),
the Vam6 amount bound to Vps41 was reduced and
ier Inc.
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 5. Effect of Vps3 and Vps8 Over-
production on Complex Composition
and Vacuole Morphology
(A) Overproduction of Vps3 leads to severe
vacuole fragmentation, whereas overproduc-
tion of Vps8 does not influence vacuole mor-
phology. BY VPS41-TAP cells with or without
VPS3 or VPS8 under the control of the GAL1
promoter were grown overnight in YPD (glu-
cose) or YPG (galactose) medium, then diluted
and grown to logarithmic phase. Cells were
stained with FM4-64 and analyzed by fluores-
cence microscopy (see Experimental Proce-
dures section for the details). Size bar = 10 mm.
(B) Overproduced Vps3 binds to Vps41. BY
VPS41-TAP cells with or without VPS3 under
the control of the GAL1 promoter were grown
overnight in YPD or YPG medium. In the morn-
ing, cells were lysed and Vps41-TAP protein
was purified as described in Figure 4C and
the Experimental Procedures section. Western
blots were decorated with antibodies to Vam6,
Vps33, Vps3, and Vac8 (as a negative control).
A model indicates the conversion of the HOPS
complex into the Vps3-Vps41-class C interme-
diate.
(C) Loss of the Vps3-Vps41-class C complex
by overproduction of Vps8. BY wild-type or
vam6D cells containing VPS41-TAP and VPS8
under the control of the GAL1 promoter were
grown overnight in YPD or YPG medium. In
the morning, cells were lysed and the Vps41-
TAP protein was purified according to the
protocol described in the Experimental Proce-
dures section and Figure 4. Proteins copurify-
ing with Vps8 were analyzed by SDS-PAGE
and western blotting using antibodies against
Vps33 and Vps3. A model indicates the con-
version triggered by Vps8 overproduction.
(D) Vacuole morphology in the presence of
overproduced Vps3. Diploid cells carrying
one allele of VPS3 under the control of the
GAL1 promoter were grown in YPD. Synthesis
of Vps3 was induced by exchanging the
medium for YPG. Cells were incubated with FM4-64 1 hr prior to each time point and incubated for 30 min in YPD (time points 0–1 hr) or YPG (all
other time points) medium (pulse), then washed and incubated for an additional 30 min in YPD or YPG (chase). Cells were analyzed by fluorescence
microscopy.
(E) Expression control of Vps3. At the same time point of microscopic analysis, an aliquot was processed for whole protein analysis. All samples were
analyzed by SDS-PAGE and western blotting using the Protein A-peroxidase (PAP) and the Vps3 antibody.
(F) Pho8 and Ape1 maturation in GAL1-VPS3 strains. Total cell extracts from pep4D, wt, and GAL1-VPS3 cells grown in galactose were prepared and
analyzed by SDS-PAGE and western blotting using antibodies to Ape1 and Pho8 (m = mature, pro = precursor).
was in part replaced by Vps3. Thus, Vps3 and Vam6 com-
pete for binding to Vps41. In agreement with this, Vam6
(Wurmser et al., 2000) and Vps3 (this study) both require
the class C subunit Vps11 for binding to Vps33 (Figure S6).
Moreover, overexpression of Vps3 leads to a processing
defect of Ape1 and Pho8, indicating defective vesicle fu-
sion at the vacuole due to loss of Vam6 from the HOPS
complex (Figure 5F). Preliminary data suggest that purified
Vps3 can displace Vam6 from the HOPS complex in vitro
(data not shown). Our data indicate that Vps3 and Vam6
occupy the same binding site on the class C complex,
which allows a shift from the HOPS complex to the
Vps3-Vps41-class C intermediate.
Deve
We subsequently asked if Vps8 would compete for the
Vps41-associated Vps3 (Figure 5C). To address this ques-
tion, we first accumulated the Vps3-Vps41-class C inter-
mediate by suppressing Vps8 expressing (lane 2) or by de-
leting VAM6 in addition (lane 2 versus 4). When Vps8 was
overproduced, the Vps3-Vps41 interaction was lost (lanes
3 and 5), most likely due to a shift of Vps3 toward the COR-
VET complex. Interestingly, overproduced Vps8 did not
affect vacuole morphology (Figure 5A), suggesting that it
cannot per se displace Vps41 from the HOPS complex,
even though it is likely that Vps8 and Vps41 occupy the
same binding site on the class C core complex. Hence,
our data suggest that the HOPS complex can convert
lopmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 745
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Figure 6. Directionality of Complex
Assembly
(A) Induction and repression of Vps8 expres-
sion. Cells with TAP-tagged VPS41 and HA-
tagged VPS8 under the control of the GAL1
promoter were grown in medium containing
raffinose to repress Vps8 expression, then ga-
lactose was added (lane 2) for the indicated
time. Afterwards, glucose was added and cells
were incubated for an additional 6–18 hr. At
each time point, an aliquot was removed from
the culture. Cells were lysed, proteins were
TCA precipitated, and analyzed by SDS-
PAGE and western blotting using antibodies
against HA and Vac8.
(B) Conversion from i-CORVET to CORVET is
unidirectional. The experiment was performed
as in (A). Glucose addition was for 6 hr. Cells
were lysed using detergent, and Vps41 was
purified using CaM beads (see Experimental
Procedures for details). A loading control was
removed from the extracts and TCA precipi-
tated. Vps41-TAP-associated proteins were
analyzed by SDS-PAGE and western blotting
using an antibody to Vps3. Vps3 and Vps41
bands were quantified by laser densitometry.
Interaction in lane 1 was set to 100%; interac-
tions in lanes 2 and 3 were 24% and 26%,
respectively.
(C) Models of tether dynamics between endo-
some and vacuole. For details see text.
into the CORVET complex via a Vps3-Vps41-class C
intermediate.
In our previous experiments, we started from Vps3-
depleted cells and followed the effect of Vps3 overpro-
duction, which resulted in vacuole fragmentation. We
wondered if overproduction of Vps3 would also affect
the vacuole morphology if it were carried out in the wild-
type background. When diploid cells containing one
copy of Vps3 under the control of the GAL1 promoter
were grown in glucose (which represses Vps3 overpro-
duction), their vacuoles appeared like wild-type vacuoles
(Figure 5D). We followed the fate of the vacuole by FM4-
64 staining over time after inducing Vps3 overproduction
by the addition of galactose to the growth medium. In-
triguingly, as soon as Vps3 overproduction was detect-
able (Figure 5E), vacuole integrity was lost (Figure 5D);
with increasing cellular Vps3 content, vacuoles became
multilobed and then fragmented completely. Since Vps3
overproduction leads to the formation of the Vps3-
Vps41-class C complex (Figure 5B), we suggest that vac-
uole fragmentation in the presence of elevated amounts of
Vps3 is due to the loss of the HOPS complex and an in-
crease of the Vps3-Vps41-class C intermediate. It is pos-
sible that tethering complex dynamics are directly linked
to endosome-lysosome morphology and identity in yeast.
Directionality of Tether Assembly
Our data indicate that the Vps3 overproduction can drive
the formation of the Vps3-Vps41-class C intermediate,
whereas Vps8 overproduction does not influence the
746 Developmental Cell 12, 739–750, May 2007 ª2007 Elsevie
HOPS complex and, thus, vacuole morphology. Our find-
ings are consistent with a linear transition from HOPS via
the Vps3-Vps41-class C intermediate to the CORVET
complex by (1) exchanging Vps3 for Vam6 (Figure 5B),
then (2) replacing Vps41 with Vps8 (Figure 5C).
To test if the transition between complexes is direc-
tional, we focused on the transition from the Vps3-
Vps41-class C to the endosomal CORVET complex. We
took advantage of the vam6D mutant containing the
VPS8 gene under the control of a galactose-inducible pro-
moter (Figures 5A and 5C). Vps8 expression was induced
by adding galactose and led to strong overproduction
(Figure 6A, lanes 1 and 2). When glucose was added to
the medium, Vps8 expression was repressed and the
protein disappeared within 6 hr (lanes 3 and 4). We then
followed the dynamics of the intermediate complex by
Vps41 pull-down. As shown before, vam6D mutants accu-
mulate the Vps3-Vps41-class C intermediate in the ab-
sence of Vps8 (Figure 6B, lane 1; 100%). Overexpression
of Vps8 leads to a loss of the intermediate complex (lane
2), presumably by replacing Vps8 with Vps41 and by gen-
erating additional CORVET complex. Subsequently, we
repressed Vps8 expression by the addition of glucose to
the medium. If the reverse reaction would be possible,
Vps41 should replace Vps8 again, thus reforming the
lost Vps3-Vps41 complex. Based on our hypothesis that
regeneration of the Vps3-Vps41-class C intermediate
can occur via the HOPS complex, this route would be
blocked in the absence of Vam6. Interestingly, we could
not recover the intermediate complex upon loss of Vps8
r Inc.
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
(Figure 6B, lane 3; 26%); the retrieved amount was as low
as during the overexpression of Vps8 (lane 2; 24%), indi-
cating that the i-CORVET was not reformed during our ob-
servation period. It is possible that the lack of reformation
is due to its assembly kinetics. Alternatively, the efficient
reformation might require the HOPS complex. In sum,
our data show that endolysosomal tethers can convert in
a directed manner, suggesting a link to organelle identity
and Rab GTPase switching (Figure 6C).
DISCUSSION
The data presented here shed light on the transition be-
tween endosome and lysosome in yeast and give valuable
insight into the maintenance of organelle identity in eu-
karyotic cells. We identify a novel endosomal tethering
complex containing Vps3 and Vps8 plus the class C pro-
teins (Vps11, Vps16, Vps18, and Vps33), which we name
the CORVET complex. This identification paved the
ground for several important findings. Our data reveal
that the CORVET and the HOPS complexes are homolo-
gous. They share the class C core as a common platform,
onto which the two additional subunits assemble: Vps41
and Vam6 for the HOPS complex, and Vps8 and Vps3
for the CORVET complex. In support of this, we show
that Vps3 is homologous to and shows similar binding
characteristics to the class C core as the HOPS-subunit
Vam6 (Figure S4). Our data indicate that CORVET is an
effector of the Rab5 homolog Vps21 and suggest that
Vps3 may act as a GEF for Vps21, since it binds Vps21
preferentially in its nucleotide-free and GDP form (Fig-
ure 3); Vps3 would therefore be equivalent to Vam6, which
has GEF activity for the yeast Rab7 homolog Ypt7
(Wurmser et al., 2000). Furthermore, as Vps8 seems to
be able to displace Vps41 from the i-CORVET (Figures 5
and 6), we suggest that the two proteins have similar
functions in each complex. Finally, we identify intermedi-
ate complexes with subunits from the CORVET and the
HOPS complex.
We present two models on how the complexes may
cooperate between endosome and lysosome. In the first
model, we assume that all four complexes can assemble
de novo and bind to different Rab-GTPases on endocytic
membranes (Figure 6C, model I). Indeed, homologs of
Vps21, called Ypt52 and Ypt53, exist and may bind to
the intermediate complexes (Singer-Kruger et al., 1994).
Each complex could promote tethering of vesicular inter-
mediates. Based on the previously identified GEF function
of Vam6 for the Rab Ypt7, we assume that the Vps8-Vam6
intermediate (intermediate toward HOPS) is required in the
direction from the endosome to the vacuole, while the
Vps41-Vps3 intermediate (i-CORVET) could be required
in retrieving material from the late endosome. In this
model, the tether composition observed during overex-
pression of Vps3 or deletion of VAM6 or VPS8 could be
the result of the de novo assembly pathway or a shift in
the balance. An alternative model (model II) links the ob-
served order of interconversion to changes in the Rab-
GTPases. Here, the CORVET complex would first convert
Deve
into the Vam6-Vps8-class C (i-HOPS) complex. By ex-
changing the Rab-GDP binding proteins Vps3 for Vam6,
the prevailing Rab GTPase could be switched from
Vps21 to Ypt7. The i-HOPS complex would in the second
stage convert into the HOPS complex by replacing Vps8
with Vps41. In a third step, the HOPS complex can change
to the Vps3-Vps41-class C intermediate (i-CORVET).
Replacing Vps3 for Vam6 leads again to a change in
Rab protein binding (Vps21 for Ypt7) due to the change
in GEF activity. To complete the postulated cycle, the
intermediate complex could convert into the CORVET
complex by replacing Vps41 with Vps8.
Our data are consistent with either model. All com-
plexes described in each model are observed in wild-
type cells, with the intermediate complexes being of low
abundance. Interestingly, the shift in complex composi-
tion is associated with morphology changes of the vacu-
ole. Only overproduction of Vps3 leads to a fragmentation
of the vacuole and an accumulation of the i-CORVET com-
plex, while Vps8 overproduction did not affect vacuole
morphology. We speculate that Vps41 and Vps8 recog-
nize Ypt7 and Vps21, respectively, in their active GTP
form. In support of this notion, Vps8 seems to interact
with Vps21 (Horazdovsky et al., 1996), in particular with
its GTP form (A. Merz, personal communication), and
binding of CORVET/Vps3 to Vps21-GTP was reduced in
the vps8D background (Figure 3C). Moreover, we show
that the CORVET complex binds Vps21-GTP and that
overexpression of the CORVET subunit Vps8 drives the
accumulation of Vps21 to dot-like structures adjacent to
the vacuole (Figure 3E). This would mean that each com-
plex consists of three parts, the class C core, a GEF (Vps3
or Vam6), and an effector protein (Vps41 and Vps8). Dur-
ing the conversion of tethers induced by overproduction,
the putative GEF is exchanged first, followed by the poten-
tial Rab effector. This order is appealing, as the GEF would
recruit the next Rab, which would bind the next effector
(Model II). On Golgi-derived vesicles, a cross-talk between
Rabs and a GEF has been observed (Ortiz et al., 2002);
here, Ypt31-GTP binds to the GEF Sec2, which then re-
cruits the Rab Sec4. In model I, each complex would exist
as a stable entity to mediate Rab binding and tethering.
Future experiments will need to address the relevance of
each model.
Our observations are consistent with the findings on the
endosome-lysosome transition in mammalian cells (Bright
et al., 2005; Luzio et al., 2000; Mullock et al., 1998; Rink
et al., 2005) and offer a molecular explanation for this pro-
cess. However, one issue is puzzling. Rink et al. (2005)
showed that hVam6/hVps39 was eluted from both
Rab5-GDP and Rab-GTP columns. In yeast, Vam6 does
not bind to the Rab5-homolog Vps21 in its GDP form
(Wurmser et al., 2000), but Vps3 does (our study). In
fact, two Vam6 homologs have been described in mam-
malian cells, TRAP-1 (Charng et al., 1998) and hVam6/
hVps39 (Caplan et al., 2001), which has also been termed
TLP (Felici et al., 2003). Both proteins seem to be ex-
pressed in all human tissues. Possibly, Vps3, which shows
higher similarity to the human Vam6 variants than yeast
lopmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 747
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
Vam6, corresponds to the human hVps39 analyzed by
Zerial and colleagues, whereas yeast Vam6 could corre-
spond to TRAP-1. In fact, depletion of hVps39 led to the
generation of a swollen hybrid organelle containing Rab5
and Rab7 (Rink et al., 2005), similar to our observations
on the deletion of VPS3 that results in a class D vacuole
phenotype (Figure 1). Therefore, the transition of endo-
some to lysosome could be mediated by directed alter-
ation of tethers on endosomal membranes, followed by
homotypic fusion with lysosomes (Figure 6C).
Our results on the modular assembly of the C com-
plexes are not without precedence. The Rabex5-Rabap-
tin5 complex consists of a Rab5 GEF and a Rab5 effector
(Horiuchi et al., 1997). Both proteins need to act together
to promote endosome fusion (Lippe et al., 2001). The
TRAPP tethering complex has been identified in two com-
positions: TRAPPI is required for ER-Golgi transport and
TRAPP II at the trans Golgi-early endosome interface
(Cai et al., 2005; Sacher et al., 1998). TRAPP I contains
GEF activity for the Rab Ypt1 (Kim et al., 2006; Wang
et al., 2000). Recently, it has been shown that the addi-
tional subunits of TRAPP II, Trs120, and Trs130 confer
GEF activity for the endosomal Rabs Ypt31/32 (Morozova
et al., 2006). Thus, TRAPPI may convert into TRAPPII to
drive vesicular transport during secretion. It is possible
that this principle also applies to other intracellular tether-
ing complexes.
In sum, our data provide an important molecular
extension to our understanding of endosome-vacuole/
lysosome biogenesis. We suggest that tethering com-
plexes control Rab GTPase switching and stability. Future
studies need to focus on the assembly and disassembly
reaction, the regulation of each complex, and their con-
nection to cargo and Rabs.
EXPERIMENTAL PROCEDURES
Yeast Strains and Molecular Biology
Strains used are listed in Table S1. Deletions and tagging of genes
were done using homologous recombination in BY4741 (MATa
his3D1 leu2D0 met15D0 ura3D0). Details on strain construction and
plasmids are available in Supplemental Data.
Microscopy
Staining of cells with the lipophilic dye FM4-64 or CMAC was per-
formed as described (LaGrassa and Ungermann, 2005). For GFP
microscopy, cells were grown logarithmically in YPD or selective
medium, collected by centrifugation, washed once with 1 ml PBS
buffer and analyzed by fluorescence microscopy. Images were ac-
quired with a Zeiss Axiovert 35 microscope equipped with an AxioCam
and a 1003 objective using filter set 10 or phase contrast, or with a
Leica DM5500 microscope and a SPOT Pursuit camera using GFP,
FM4-64 by phase contrast or DIC filters. Pictures were processed
using Adobe Photoshop 7.0.
Yeast Cell Lysis
Cells were fractionated essentially as described (LaGrassa and Unger-
mann, 2005). For details see Supplemental Data.
CaM Pull-Down
A cleared detergent extract of total cell lysate was generated as above.
A fraction (5%) of the total protein amount was removed as a loading
748 Developmental Cell 12, 739–750, May 2007 ª2007 Elsevie
control (followed by TCA precipitation), and the rest was loaded onto
80 ml of prewashed CaM beads (GE Healthcare). Samples were incu-
bated for 2 hr at 4C in the presence of 2 mM CaCl2. Beads were
washed three times for 10 min with buffer (0.2 M sorbitol, 150 mM
KCl, 20 mM HEPES/KOH, [pH 6.8], 2 mM CaCl2) with decreasing Triton
X-100 concentrations (0.5%, 0.1%, 0.025%), and proteins were eluted
by addition of 2 mM EGTA followed by a 20 min incubation at 30C.
Beads were reisolated, the eluate was transferred to a fresh tube,
TCA precipitated, and analyzed by SDS-PAGE and western blotting.
Blots were analyzed using the primary antibody indicated, and sec-
ondary antibodies coupled to dyes or horseradish peroxidase. Detec-
tion was performed using a LICOR-ODYSSEY system or by standard
enhanced chemiluminescence.
TAP-Tag Protein Purification
TAP-tag protein purification was performed as described in Rigaut
et al. (1999) using the following buffer: 50 mM HEPES/KOH, (pH 7.4),
300 mM NaCl, 0.15% NP-40 (Igepal CA-630; Sigma-Aldrich), and
1.5 mM MgCl2.
Gel Filtration
Protein complexes were purified as described in TAP-tag protein puri-
fication protocol omitting the CaM-bead purification step. The TEV
eluate was centrifuged for 10 min at 20,000 3 g to pellet insoluble
material. The supernatant was applied to a Superose 6 10/300 column,
connected to an AKTA-FPLC-System (GE Healthcare), which had
been equilibrated with two column volumes of TAP-purification buffer.
The flow rate was set to 0.3 ml/min, and 24 1 ml fractions were col-
lected. For analysis, fractions 6–19 were TCA precipitated and loaded
onto a 4%–12% SDS-PAGE gradient gel (NuPAGE, Invitrogen).
GSH Pull-Down
GST fusion proteins (400 mg per sample) were bound to GSH beads
and washed three times with 500 ml 20 mM HEPES/KOH (pH 7.4),
100 mM NaCl, 10 mM EDTA, 0.1%TX100. Beads were resuspended
in 20 mM HEPES/KOH (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 0.1%
TX100, and 0.5 mM GDP, GTPgS or no nucleotide, and incubated
for 1 hr at 4C. Purified CORVET (fractions 10 and 11) was prepared
from the GAL1-TAP-VPS3 overexpression strain as described above,
and 1 ml was added to each sample. Alternatively, lysates from the
indicated strains were prepared by glass bead lysis from 3 l of cells
in 20 mM HEPS/KOH (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 0.1%
TX100, centrifuged (1 hr, 100,000 3 g, 4C), concentrated to 2 ml using
an Amicon Ultra Centrifugal Filter Device (MWCO 10,000), and added
to the prebound Rab GTPases. Beads were incubated for 1 hr at 4C
on a rotating wheel, washed four times with decreasing TX100 con-
centrations, and eluted by incubating beads in 20 mM HEPES/KOH
(pH 7.4), 1.5 M NaCl, 20 mM EDTA, 0.025% TX-100 for 20 min at
room temperature. Eluates were TCA precipitated and analyzed by
SDS-PAGE and western blotting.
Supplemental Data
Supplemental Data include supplemental Experimental Procedures
and references, six supplemental figures, and one supplemental table
and are available at http://www.developmentalcell.com/cgi/content/
full/12/5/739/DC1/.
ACKNOWLEDGMENTS
We thank Alexey Merz for communicating results prior to publication;
Tom Stevens for kindly sharing the Vps3 antibody with us; Francis
Barr, Scott Emr, Alexey Rak, Roger Goody, and Bruno Antonny for dis-
cussions; Michael Knop for plasmids; Christoph Meiringer for help with
microscopy; Angela Perz and Gabriela Muller for expert technical as-
sistance; and all members of the Ungermann group for support and
discussion. This work was supported by the DFG (UN111/3-1; Heisen-
berg program), the SFB 638 and 431, the EMBO Young Investigator
Program, and the Fonds der Chemischen Industrie. C.U. is supported
r Inc.
Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
by the Hans-Muhlenhoff foundation. K.P. has been a recipient of a sti-
pend of the Landesgraduiertenforderung Baden-Wurttemberg. Part of
this work has been performed at the University of Heidelberg.
Received: September 25, 2006
Revised: December 27, 2006
Accepted: March 8, 2007
Published: May 7, 2007
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Supplemental Data
The CORVET Tethering Complex Interacts
with the Yeast Rab5 Homolog Vps21
and Is Involved in EndoLysosomal Biogenesis Karolina Peplowska, Daniel F. Markgraf, Clemens W. Ostrowicz, Gert Bange, and Christian Ungermann
Supplemental Experimental Procedures
Yeast strains and plasmids
To generate plasmids carrying GFPtagged proteins, a BamH1BglII fragment from pBSeGFP
(provided by E. Hurt, Heidelberg, Germany) was inserted into a BamH1 site of the indicated plasmid.
Plasmids were either genomically integrated by cutting with Bsp119I (pRS406NOP1prGFPVam3),
or maintained within cells by growth on selective medium (pRS415NOP1prGFPVPS41, pRS416
NOP1prGFPYCK3, pRS426NOP1prVAC8GFP). Cterminal tagging of VPS3 and VPS8 in the
indicated strains was done by integrating a PCRamplified region coding for the TAPtag (pYM13) or
the GFPtag (pYM12) and the kanamycin marker (kindly provided by M. Knop, Heidelberg, Germany)
via homologous recombination. TAPtagging of VPS41 or VPS3 was done similarly, using pBS1539 as
a template (Puig et al., 1998). VPS3, VPS8, VPS9, or the Rab GTPases VPS21, YPT7 and YPT1 were
placed under the control of the GAL1promoter using PCR fragments containing flanking regions of
the respective genes amplified from pFA6aHIS3MX6GAL1pr (VPS3), pFA6aHIS3MX6GAL1pr
3xHA (VPS8), pBS1761TRPGAL1prTAP (VPS3, VPS9), or pFA6kanMX6GAL1prGST (VPS21,
YPT7, YPT1) (Longtine et al., 1998). PHO8 and VPS21 were genomically tagged at the Nterminus
using a URA3PHO5prGFPMyc cassette, amplified from plasmid pGL (a gift from S. Munro, MRC,
Cambridge, UK; Levine and Munro, 2001). VPS21, YPT7, and YPT1 were cloned into pGEX4T3 or
pGEX2T (GE Healthscience) and purified according to the manufacturer.
2
Yeast cell lysis
After overnight growth in rich medium containing 2% glucose (YPD) or 2% galactose (YPG), cell
cultures were diluted to OD600=0.5 and incubated for 2 hours in 30°C. Cells (30 OD600 units) were
collected, washed once with DTT buffer (10 mM DTT, 0.1 M Tris/HCl pH 9.4), resuspended in 1 ml of
DTT buffer and incubated for 10 minutes in 30°C. They were then centrifuged (2 min at 4620g),
resuspended in 300 µl of spheroplasting buffer (0.16x YPD, 50 µM KPi buffer, pH 7.4, 0.6 M sorbitol),
and incubated for 20 min at 30°C in the presence of lyticase. Cells were centrifuged for 3 min at 1530g,
the pellet was resuspended in 1ml of lysis buffer (0.2 M sorbitol, 150 mM KCl, 20 mM HEPES/KOH,
pH 6.8, 1 mM DTT, 1 mM PMSF, 1xPIC) supplemented with 6 µl of 0.4 mg/ml DEAEdextran, and
incubated for 5 minutes on ice. Samples were briefly heatshocked (2 min/30°C), and unlysed cells
were removed by centrifugation at 300g for 3 min. The cell lysate was used in further experiments
(pulldowns, subcellular fractionations).
GST pull down
Cells were grown overnight in the presence of galactose to overproduce GSTRab protein
(Vps21, Ypt7 or Ypt1) together with TAPtagged Vps3 and Vps9. 200 ODs of cells were collected,
washed once with 1 ml of buffer A (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2) and lysed
with glass beads in a presence of 300 µl of buffer A containing1xPIC and 1 mM PMSF. Lysis was
repeated twice and each time 300 µl of lysate were collected. The lysate (25 mg) was supplemented
with 0.5% Triton X100, centrifuged (30 min, 100,000g, 4°C), and then loaded onto 50 µl equilibrated
glutathione (GSH) beads. An aliquot of the lysate (0.1%) was removed as a loading control. Beads
were incubated at 4°C for 1.5 hour and then washed extensively (2 x 15 min with buffer A + 0.1%
Triton X100 and 2 x 15 min with buffer A + 0.025% Triton X100). Proteins were eluted by
3
incubating the beads for 20 min at room temperature in 600 µl elution buffer (20 mM Tris/HCl, pH 7.4,
1.5 M NaCl, 20 mM EDTA), TCAprecipitated, and analyzed by using 7.5% SDSPAGE gels and
Western blotting.
References:
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCRbased gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953961.
Puig, O., Rutz, B., Luukkonen, B. G., KandelsLewis, S., BragadoNilsson, E., and Seraphin, B. (1998). New constructs and strategies for efficient PCRbased gene manipulations in yeast. Yeast 14, 11391146.
4
Figures
Figure S1. Characterization of vps3∆ mutants.
(A) Osmotic stress response. Logarithmically grown wt and vps3∆ cells were stained with FM464,
reisolated, incubated in YPD medium containing 0.4 M NaCl, and analyzed by fluorescence
microscopy after 10 and 60 minutes. Control cells did not receive salt. Quantification of at least 200
cells per condition is shown.
(B) Vacuole morphology of vps3∆ cells. BY4741 cells (wt,vps3∆ or vps8∆) were grown
logarithmically in YPD medium. To visualize vacuoles, cells were incubated for 30 minutes (pulse)
with 25 µM CMAC dye (7amino4chloromethylcoumarin) , washed, and incubated for another 30
minutes (chase) in fresh YPD before being analyzed by fluorescence microscopy.
Figure S2. Characterization of the Vps3 protein.
Localization of the Vps3 protein. BY4741 cells expressing Vps3GFP were analyzed by fluorescence
microscopy.
Figure S3. Alignment of Vps3 and Vam6 proteins from yeast and human.
The alignment was done with Jalview (Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (2004),
"The Jalview Java Alignment Editor", Bioinformatics, 12, 4267.).
5
Figure S4: Cooverexpression of Vps3 and Vps21.
Cells cooverexpressing the indicated RabGTPase in the wt or GDPlocked form (S21N) and TAP
tagged Vps3 (3) or Vps9 (9) were grown overnight and processed for the GSH pulldown as described
in Experimental Procedures. Specifically bound proteins were eluted and analyzed by SDSPAGE and
Western blotting. The lower band in lane 3 is most likely a degradation product of Vps3 that seems to
bind efficiently to Vps21S21N. Note that Vps3 was overproduced more strongly in the absence of any
GTPase (lane 6), similarly Vps9 showed stronger expression (lane 10). A lower exposure of the load is
shown in the bottom right panel.
(C) RabGTPase present on the remaining GSH beads. Beads were boiled in SDSsample buffer and
analyzed as above. Note that Vps21S21N is poorly recovered on GSH beads, potentially due to its
decreased stability caused by the mutation.
(D) Expression of the fusion proteins in vivo. Whole cell extracts were prepared from galactose
induced cultures as described in Experimental Procedures, proteins were resolved by SDSPAGE and
analyzed by Western blotting using antiGST and ProteinAperoxidase coupled antibodies.
Figure S5. Accumulation of GFPVps21 upon Vps8 overexpression.
The experiment was done as in Figure 3F, and shows representative fields. Size bar is 10 µm.
Figure S6. Vps3 interaction with the Class C complex.
Purification of Vps3TAP from vps11∆ cells was done as described above. Eluted proteins were
analyzed by SDSPAGE and Western blotting as described before.
6
7
8
9
10
11
12
Table S1. Strains used in this study
Strain Genotype Reference CUY1 BJ3505;MATa pep4::HIS3 prb1∆1.6R HIS3 lys2208 trp1∆101 ura352 gal2 can Haas et al., 1994 CUY476 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Euroscarf library CUY473 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps8∆::kanMX Euroscarf library CUY765 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam3∆::kanMX This study CUY865 CUY476; pRS426NOP1prVAC8GFP Subramanian et al., 2006 CUY887 BY4741;MATa pRS415NOP1prGFPVPS41 This study CUY959 CUY476; PHO8::HIS5PHO5prGFPMYC LaGrassa and Ungermann, 2005 CUY1014 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps3∆::kanMX Euroscarf library CUY1616 BJ3505; VPS3::TAPkanMX This study CUY1792 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS8::TAPkanMX This study CUY1794 CUY476; VPS3::GFPkanMX This study CUY1795 CUY476; VPS3::TAPURA3 This study CUY1796 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps41∆::kanMX VPS3::TAPURA3 This study CUY1797 CUY473; VPS3::TAPURA3 This study CUY1798 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam6∆::kanMX VPS3::TAPURA3 This study CUY1799 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps33∆::kanMX VPS3::TAPURA3 This study CUY1800 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS41::TAPURA3 This study CUY1801 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam6∆::kanMX VPS41::TAPURA3 This study CUY1802 CUY1014; VPS41::TAPURA3 This study CUY1803 CUY473; VPS41::TAPURA3 This study CUY1804 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps33∆::kanMX VPS3::TAPURA3 This study CUY1805 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 ypt7∆::kanMX VPS3::TAPURA3 This study CUY1806 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps21∆::kanMX VPS3::TAPURA3 This study CUY1819 CUY473; VPS21::URA3PHO5prGFP This study CUY1820 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vam6∆::kanMX VPS21::GFPURA3 This study CUY1826 CUY1014; VPS21:: URA3PHO5pr GFPMYC This study CUY1836 CUY1014; PHO8::URA3PHO5prGFPMYC This study CUY1837 CUY1014; pRS426NOP1prGFPVAC8 This study CUY1838 CUY476; URA3::pRS406NOP1prGFPVAM3 This study CUY1839 CUY1014; URA3::pRS406NOP1prGFPVAM3 This study CUY1841 CUY476; pRS416NOP1prGFPYCK3 (URA3) This study CUY1842 CUY1014; pRS416NOP1prGFPYCK3 (URA3) This study CUY1845 CUY1794; vps8∆::URA3 This study CUY1847 CUY1800; VPS3::HIS3GAL1pr This study CUY1849 CUY1014; pRS415NOP1prGFPVPS41 (LEU2) This study CUY1850 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps8∆::kanMX VAM3::URA3 This study CUY1877 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS8::TAPkanMX vps33∆::URA3 This study CUY1878 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS3::TAPURA3 vps11∆::kanMX This study CUY1883 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS41::TAPkanMX VPS8::HIS3GAL1pr3HA This study CUY1895 BY4733;MATalpha; his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS3::TRP1GAL1prTAP This study CUY1897 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vam6∆::kanMX VPS8::HIS3GAL1pr3HA
VPS41::TAPURA3 This study
CUY1915 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS3::kanMXGAL1pr This study CUY1936 CUY1895; VPS21::kanMXGAL1prGST This study CUY1949 CUY1895; VPS21 S21N::kanMXGAL1prGST This study CUY1948 CUY1895; YPT1::kanMXGAL1prGST This study CUY1952 BY4719;MATa trp1∆63 ura3∆0 VPSs8::kanMX VPS21::URAPHO5prGFP pV2RFPYPT7 (TRP1) This study CUY1960 BY4719;MATa trp1∆63 ura3∆0 vps8∆::kanMX VPS21::URAPHO5prGFP pV2RFPYTP7(TRP1) This study CUY1967 BY4719;MATa trp1∆63 ura3∆0 VPS21::URA3PHO5prGFP This study CUY1969 BY4733;MATalpha; his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS9::TRP1GAL1TAP
VPS21::kanMXGAL1prGST This study
CUY1972 BY4719;MATa trp1∆63 ura3∆0 VPS21::URA3PHO5prGFP pV2RFPYPT7 (TRP1) This study CUY2251 BY4733;MATalpha his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VAM6::kanMXGAL1pr
VPS21:: URA3PHO5prGFP This study
CUY2252 CUY1915; VPS21::URA3 PHO5prGFP This study CUY2253 BY4733;MATalpha his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS8::HIS3GAL1pr3HA
VPS21:: URA3PHO5prGFP This study
CUY2359 BY4719;MATa trp1∆63 ura3∆0 vps8∆::kanMX VPS3::TRP1GAL1prTAP This study CUY2278 CUY1826; VPS8::HIS3GAL1pr3HA This study