From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038
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ABSTRACT |
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Vacuolar protein sorting (vps)
mutants of Saccharomyces cerevisiae missort and secrete
vacuolar hydrolases. The gene affected in one of these mutants,
VPS21, encodes a member of the Sec4/Ypt/Rab family of small
GTPases. Rab proteins play an essential role in vesicle-mediated
protein transport. Using both yeast two-hybrid assays and chemical
cross-linking, we have identified another VPS gene product,
Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N
that binds GDP but not GTP. In vitro purified Vps9p was
found to stimulate GDP release from Vps21p in a
dose-dependent manner. Vps9p also stimulated GTP
association as a result of facilitated GDP release. However, Vps9p did
not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab
protein involved in vacuolar protein transport. Rab5, but not Ypt7p was
responsive to Vps9p, which indicates that Vps9p recognizes sequence
variation among Rab proteins. We conclude that Vps9p is a novel guanine
nucleotide exchange factor that is specific for Vps21p/Rab5. Since
there are no obvious Vps9p sequence homologues in yeast, Vps9p may also
possess unique regulatory functions required for vacuolar protein transport.
Vesicle-mediated protein transport is responsible for executing
many intracellular protein trafficking events (1). This process is
mediated by complex machinery that is highly conserved from yeast to
cells of higher eukaryotes (2, 3). Members of the Sec4/Ypt/Rab family
of small GTP-binding proteins are an integral part of this conserved
machinery and are thought to participate in the targeting and/or fusion
of transport vesicles with the appropriate target membrane (4, 5).
Although the exact function(s) of Rab proteins is unknown, vesicle
targeting events have been coupled to the cycling of Rab proteins
between their GTP-bound and GDP-bound states, leading to the following
model (4, 5). GTP-bound Rab proteins associate with transport vesicles
derived from the donor compartment. Transport vesicles with this form of the Rab protein are competent for targeting to the acceptor organelle (6). At the acceptor organelle, a GTPase-activating protein
or GAP may act on the Rab to stimulate the hydrolysis of Rab bound GTP
to GDP (7). The GDP-bound Rab is then recycled back to the vesicle
donor membrane in a complex with GDP-dissociation inhibitor
(GDI)1 (for review, see Ref.
8). Reloading Rab proteins with GTP is thought to involve two steps. In
the first step, the Rab protein is dissociated from GDI by a GDI
dissociation factor (GDF) (9). Once separated from GDI, the Rab protein
is now accessible to the activity of a guanine nucleotide exchange
factor (GEF) that facilitates the exchange of GDP for GTP. The Rab
protein in its GTP-bound form is now capable of participating in
another round of vesicle targeting and fusion.
An important regulatory step within the Rab cycle is at the stage of
guanine nucleotide exchange. Several GEFs have been described for
members of the Sec4/Ypt/Rab family of small GTP-binding proteins. Novick and colleagues (10) have demonstrated that Sec2p possesses guanine nucleotide exchange activity for Sec4p. Sec4p is involved in
vesicle-mediated transport of secretory proteins from the yeast Golgi
to the plasma membrane (11, 12). In addition, a GEF has been purified
from rat brain that shows specificity for Rabs 3A, 3C, and 3D (13).
Interestingly, primary amino acid sequence comparisons fail to show any
obvious sequence similarity among the GEFs that stimulate guanine
nucleotide exchange of Sec4/Ypt/Rab GTP-binding proteins; indicating
that each of these GEFs function in distinct vesicular transport pathways.
Vesicle-mediated transport plays an important role in the localization
of proteins to the lysosome-like vacuole in yeast. Most vacuolar
proteins follow the initial stages of the secretory pathway until they
reach a late Golgi compartment. There, vacuolar proteins are actively
sorted away from secretory proteins, packaged into transport vesicles
and delivered to the vacuole via a prevacuolar endosome (for review,
see Ref. 14). Genetic studies of the vacuolar protein sorting (vps)
pathway have identified a large number of mutant yeast strains that
missort and secrete vacuolar proteins (15-18). These vps
mutants (vacuolar protein sorting
defective) fall into over 40 complementation groups. One group of
vps mutants (termed class D) (19) appears to affect a single
stage in the vps pathway, the transport of proteins from the Golgi to
the prevacuolar endosome (20). Many of gene products affected in the
class D vps mutants have been implicated specifically in the
targeting and/or fusion of Golgi-derived transport vesicles and several are members of highly conserved protein families (21-28). One of these, Vps21p, is a small GTP-binding protein of the Sec4/Ypt/Rab family. VPS21 was originally identified by complementation
of the vacuolar protein missorting phenotype associated with
vps21 mutant cells (25) and by its sequence similarity with
mammalian Rab5 (29). A detailed mutational analysis has demonstrated
that GTP-binding and membrane association are required for Vps21p
function (25). In addition, cells that lack Vps21p not only missort
vacuolar hydrolases, but also accumulate 40-50-nm vesicles (25, 29), indicating a role for Vps21p in vesicle targeting and or fusion events.
To better understand the role of the Vps21 GTP-binding protein in
vesicle targeting, we undertook a study to identify modulators of
Vps21p function among the gene products affected in the class D
vps mutants. Using in vitro and in
vivo techniques, a physical interaction was uncovered between
Vps21p and Vps9p. This interaction is potentiated when a mutant form of
Vps21p is used that possesses a higher affinity for GDP than GTP. In
addition, we show that Vps9p is a GEF that stimulates the intrinsic
guanine nucleotide exchange rate of Vps21p. The guanine nucleotide
exchange activity of Vps9p is specific for Vps21p and its mammalian
sequence homologue, Rab5.
Strains, Media, and Other Reagents--
The Saccharomyces
cerevisiae strains used in this study were: L40 (MATa
trp1 leu2 his3
LYS2::(lexAop)4-HIS3
URA3::(lexAop)8-lacZ (30),
SEY6210 (MATa leu2-3, 112 ura3-52 his3- Plasmid Construction--
To create the Vps21p, Vps21p-S21N
two-hybrid baits, and the Vps9p two-hybrid prey, the coding sequences
of these genes were amplified by PCR using pGBY21-5, pBHY21-11 (25),
and pPS91 (27), respectively, as templates and oligonucleotides that
resulted in PCR products which contained 5' BamHI and 3'
SalI linkers. These products were ligated into either pVJL11
(36) to create the Vps21p and Vps21p-S21N baits (pGT21-1 and pGT21-2,
respectively) or pGADGH (37) to create the Vps9p prey, GT9-1.
Expression of these fusion proteins in the L40 strain was confirmed by
immunoblotting. To create CEN and 2-µm yeast expression
plasmids encoding C-terminal HA-tagged Vps9p, a SmaI site
was created at the 3' end of the VPS9 coding sequence
eliminating the stop codon using PCR mutagenesis. This amplified
product was digested with SmaI and HindIII and inserted into the same sites of pRS416 to create pGT9-3. A
SmaI-EcoRV fragment containing the coding
sequence for the HA-epitope from plasmid YEp352-HA was inserted into
the SmaI site of pGT9-3 to create pGT9HA-1. The
EcoRV-HindIII fragment of pGT9HA-1 was subcloned into pRS426 (38) to create the 2-µm plasmid, pGT9HA-2. To create Vps21p and Vps21p-S21N E. coli expression constructs, the
VPS21 and VPS21-S21N coding sequences were
amplified by PCR from pGBY21-2 (25) and pBHY21-11 (25). These
products were cloned into the E. coli expression vector,
pKK223-3 (Pharmacia Biotech) to create the Vps21p and the Vps21p-S21N
expression constructs pBHY21-30 and pBHY21-76. pQEVPS9, which
contains the VPS9 gene modified with a
NH2-terminal hexahistidine-coding sequence, was constructed in the same manner as pGT9-1 except pQE31 (QIAGEN, Inc.) was used. The
NH2-terminal sequence of the His6-Vps9p is
M-R-G-S-(H)6-T-D-P-Vps9p. A His6-Vps9p yeast
expression construct, pEMBL-VPS9 was constructed by cloning the
(His)6-VPS9 gene from pQEVPS9 into pEMBLye30/2 (39). The E. coli Ypt7p expression construct was created by amplifying the YPT7 gene from pBS-YPT7 (40) and cloning the PCR product into pKK223-3 resulting in pKKYPT7. The
vps21::NEOR construct was generated by
first subcloning the SmaI/SpeI kan MX4 module
from pFA6-kanMX4 (41) into pBluescript II (Stratagene) to create
pBS-NEO. An EcoRV fragment containing the kanMX4 module from
pBS-NEO was inserted into the Klenow enzyme-treated
SalI/BglII sites of pBHY21-18 to create plasmid
pBHY21-78.
Yeast Two-hybrid Assay--
L40 yeast cells (30) were
transformed with pGT21-1, pGT21-2, or pVJL11 (36) and either pGT9-1 or
pGADGH (37). Transformants were selected and streaked onto SM plates
lacking tryptophan, leucine, and histidine. Protein Cross-linking--
GTY1 (vps9 Purification of Vps21p and Ypt7p--
Wild-type Vps21p and
Vps21p-S21N were purified from E. coli CW2642 carrying
pBH21-30 or pBH21-76, respectively. Recombinant proteins were induced
with 1 mM isopropyl- Purification of (His)6-Vps9p--
M15 E. coli cells carrying pREP4 and pQEVPS9 were grown and induced as
described for the Vps21p purification. Cells were harvested, washed,
lysed, and (His)6-Vps9p was purified from the crude cell extract using Ni-NTA agarose as described by the manufacturer (Qiagen).
(His)6-Vps9p was eluted from the Ni-NTA agarose,
concentrated, dialyzed against buffer B (50 mM Tris-HCl, pH
7.5, 10% glycerol), and loaded onto a Q2 column. Proteins were eluted
with a 0 to 300 mM linear NaCl gradient in buffer B.
Guanine Nucleotide Binding Assay--
Wild-type or Vps21p-S21N
was incubated in 50 µl of 50 mM Tris-HCl, pH 7.5, 1 mM MgSO4, 1 mM DTT, 3 mM EDTA, 50 µM [3H]GDP
(2.6 × 104 cpm/µl), or [ Guanine Nucleotide Displacement and Exchange
Assays--
Displacement was monitored by incubating preloaded
[3H]GDP or [ Immunoprecipitation of CPY--
Yeast cells were grown, labeled
with [35S]methionine and cysteine and subjected to
immunoprecipitation as described (25).
S21N Mutant Form of Vps21p Binds GDP but Not GTP--
The activity
of small GTP-binding proteins of the Ras family is highly regulated.
This regulation is carried out by a growing number of factors that
modulate or stabilize the guanine nucleotide associated with the
appropriate GTPase. In this study, we undertook a search for factors
that associate with Vps21p in its GDP-bound form and in doing so hoped
to uncover the factor(s) responsible for exchanging GDP for GTP. An
extensive characterization of the nucleotide binding capabilities of
mutant Ras proteins (44) have identified a number of amino acid
alterations that result in proteins that show great preferences for
binding GDP or GTP. One of these, Ras N17, has been shown to bind GDP
with a 20-40-fold higher affinity than GTP (44). The equivalent mutant
in Vps21p (S21N) has been constructed and was shown to elicit defects
in the vacuolar protein sorting pathway, indicating the importance of
GTP binding for Vps21p function in vivo (25). To
characterize the guanine nucleotide binding preferences of recombinant
wild-type Vps21p and the Vps21p-S21N mutant form of the protein, these
proteins were purified from E. coli as described under
"Experimental Procedures" and their abilities to bind GDP and GTP
were examined. The estimated purity of these proteins was 75 and 80%
for the wild-type and Vps21p-S21N, respectively (Fig.
1A), and no other GTP-binding proteins were detected in these fractions by [32P]GTP
blot analysis.2 Purified
wild-type and S21N-Vps21p were incubated with [3H]GDP or
[32P]GTP in the presence of EDTA. Endogenous nucleotides
bound to Vps21p rapidly exchanged with radiolabeled nucleotides under
these conditions, reaching maximum binding within 30 min.2
As shown in Fig. 1B, Vps21p-S21N bound substantially more
GDP (26 pmol) than GTP (<2 pmol). Whereas, wild-type Vps21p bound slightly more GTP (32 pmol) than GDP (24 pmol). These results demonstrate that Vps21p-S21N, like the Ras N17 protein, preferentially binds GDP.
Vps21p and Vps9p Physically Interact--
vps21 mutants
fall into the class D vps morphology group. This group
shares a unique subset of phenotypes including, vacuolar protein
sorting defects, enlarged vacuolar structures, a temperature-sensitive growth phenotype, as well as defects in mother to daughter vacuole segregation and vacuole acidification. Previous studies have shown that
several of the gene products affected in these mutants likely function
at the same stage in the vacuolar protein sorting pathway (20, 45) and
two (Vps15p and Vps34p) physically interact (46). The yeast two-hybrid
system was used to uncover potential interactions between Vps21p and
gene products affected in other class D vps mutants. LexA
gene fusions were constructed that contained wild-type or mutant S21N
VPS21 coding sequences. A second set of gene fusions was
constructed between the activation domain of Gal4p (Gal4AD) and other
class D VPS gene products. These constructs were used to
transform a yeast strain (L40) that contained the HIS3 and lacZ reporter gene constructs under control the
LexA promoter. As shown in Fig.
2, L40 yeast that expressed both the
LexA-Vps21p-S21N fusion and the Gal4AD-Vps9p fusion were prototrophic
for histidine. On the contrary, L40 yeast that expressed both the
LexA-Vps21p wild-type fusion and the Gal4AD-Vps9p fusion, or the
LexA-Vps21p-S21N or Gal4AD-Vps9p fusions alone were not prototrophic
for histidine (Fig. 2A). All strains tested were able to
grow on synthetic media that contained histidine (Fig.
2B).
A second reporter system was utilized to score an interaction between
the LexA-Vps21p-S21N fusion and the Gal4AD-Vps9p fusion. The same five
strains tested in Fig. 2, A and B, were patched onto an agar plate containing synthetic media (+ histidine). The yeast
cells were transferred to nitrocellulose filters, lysed, and the
presence of
Cross-linking studies were used to confirm the Vps21p-Vps9p two-hybrid
results and to examine if the two-hybrid interaction accurately
represented an in vivo phenomenon. Spheroplasts generated from cells (GTY1; vps21
The levels of Vps21p in the cell lysates were also determined and
compared with the amount of Vps9HAp that was cross-linked. Strains
expressing wild-type Vps21p from a high copy number plasmid produced
approximately 7-fold more Vps21p (Fig. 3B, lanes 8 and 9) than strains expressing Vps21p-S21N from a low copy
number plasmid (Fig. 3B, lanes 4-7) (these immunoblots were
exposed to film for different amounts of time as indicated in the
figure legend). However, the amount of Vps9HAp that was cross-linked to
Vps21p-S21N was approximately 10-fold more than what was cross-linked to wild-type Vps21p despite the fact that wild-type Vps21p was expressed from a high copy number vector (compare Fig. 3A, lanes 6 and 8). It should be noted that when Vps21p was
overexpressed, a slower migrating form of the protein (23 kDa) is seen.
This larger form represents unprenylated Vps21p (25). These results indicate that Vps9HAp has a higher affinity for Vps21p-S21N than wild-type Vps21p. These observations are consistent with the two-hybrid results which indicated that Vps9p interacts more strongly with Vps21p-S21N.
Vps9p Stimulates Nucleotide Exchange Activity of Vps21p in
Vitro--
To facilitate the exchange of GDP for GTP, guanine
nucleotide exchange factors are likely to first recognize and then
associate with Rab proteins in their GDP-bound state. The preferential
association of Vps9p with Vps21p in its GDP-bound state (S21N mutant)
suggested that Vps9p may function as a guanine nucleotide exchange
factor. To test this possibility, guanine nucleotide displacement
assays were carried out with purified Vps21p in the presence of
wild-type cell extracts or cell extracts from a strain that
overexpressed Vps9p approximately 14-fold. Purified Vps21p was
preloaded with [3H]GDP and then diluted into an exchange
buffer that contained a 130-fold excess of unlabeled GDP. Because of
the intrinsic nucleotide exchange activity of Vps21p (Fig.
4, triangles), pre-bound
radioactive GDP was slowly exchanged for unlabeled GDP, resulting in a
decrease in Vps21p-bound 3H counts with increasing time.
This intrinsic exchange was not significantly affected by adding cell
extract from wild-type yeast (Fig. 4, circles). However, an
extract from cells overproducing Vps9p stimulated this intrinsic
exchange 1.8-fold (Fig. 4, squares). This result indicated
that a cell lysate containing an increased amount of Vps9p enhanced the
intrinsic guanine nucleotide exchange rate of Vps21p.
In order to demonstrate that this stimulatory effect was directly due
to the presence of Vps9p, purified recombinant Vps9p was used in the
exchange assay. Both bacterial and yeast expression plasmids were
constructed that encoded Vps9p with an amino-terminal hexahistidine tag
((His)6-Vps9p). To determine if the His-tagged version of
Vps9p was functional in vivo, the yeast expression plasmid
was introduced into a strain that lacked Vps9p (CBY20, vps9
An E. coli strain was transformed with the bacterial
expression construct and used to purify (His)6-Vps9p.
Although the majority of the overproduced (His)6-Vps9p
formed inclusion bodies in E. coli, we were able to purify
(His)6-Vps9p from soluble fractions to >90% purity (Fig.
5B). When purified Vps9p was added to the displacement
assay, release of pre-bound [3H]GDP was stimulated in a
dose-dependent manner (Fig.
6A). In addition, Vps9p acted
catalytically. At a molar ratio of 1:4 (Vps9p:Vps21p), Vps9p was able
to significantly stimulate nucleotide displacement (Fig. 6A,
Vps9p Does Not Stimulate GTP Release from Vps21p--
It has been
shown that nucleotide exchange factors can associate with both GDP- and
GTP-bound GTPases and stimulate nucleotide release by stabilizing the
nucleotide-free form of the GTPases (21, 47-49). In these cases,
release of either bound GDP or GTP can be stimulated by nucleotide
exchange factors. To examine whether Vps9p behaves similarly, the
displacement assay was carried out using Vps21p preloaded with either
[3H]GDP or [ Vps9p Stimulates Nucleotide Exchange of Vps21p and Rab5 but Not of
Ypt7p--
Rab nucleotide exchange factors have been shown to act on
distinct sets of Rab proteins (10, 13, 49-51). In order to determine if Vps9p recognized structural variations among Rab proteins, the
ability of Vps9p to stimulate the intrinsic nucleotide exchange of
mammalian Rab5 and yeast Ypt7p was examined. Each GTPase was preloaded
with [3H]GDP and their intrinsic guanine nucleotide
exchange activities were determined using the [3H]GDP
release assay described in the legend to Fig. 4. As seen in Fig.
8, each GTPase possessed intrinsic
exchange activity of different magnitudes (Fig. 8, filled
triangles). Addition of Vps9p did not alter the intrinsic
nucleotide exchange rate of Ypt7p (Fig. 8C). However, the
presence of Vps9p stimulated the intrinsic nucleotide exchange rates of
Vps21p (Fig. 8A) and mammalian Rab5 (Fig. 8B).
These results demonstrate that Vps9p recognizes sequence variations
among different Rab proteins.
This study describes physical and functional interactions between
Vps9p and a Rab protein involved in vacuolar protein transport, Vps21p.
The yeast two-hybrid system was used to uncover an interaction between
Vps9p and a mutant Vps21p that binds GDP but not GTP. Protein
cross-linking confirmed this interaction. Purified recombinant Vps9p
stimulated GDP release and GTP association of Vps21p. Vps9p also
stimulated GDP release of the Vps21p mammalian sequence homologue, Rab5
in vitro. These data demonstrate that Vps9p possesses all the hallmarks of a guanine nucleotide exchange factor. Several other
observations indicate that the guanine nucleotide exchange activity of
Vps9p is physiologically relevant in vesicle-mediated protein transport
to the yeast vacuole. First, deletion of the VPS9 gene
causes severe defects in vacuolar protein transport and leads to the
accumulation of 40-50 nm vesicles (27); these phenotypes are very
similar to those of vps21 Several guanine nucleotide exchange factors for Rab proteins have been
described. These include Rab3 GEP (13) and yeast Sec2p (10) which have
been shown to be specific guanine nucleotide exchange factors for
Rab3A, C, D, and Sec4p, respectively. Surprisingly, Vps9p does not
share significant sequence similarity to Rab3 GEP or Sec2p, indicating
that this group of proteins is quite diverse. However, a mammalian
Vps9p sequence homologue, Rabex5, has been recently been identified as
a nucleotide exchange factor for Rab5 (52). Since Rab5 is a sequence
homologue of Vps21p, it is likely that Vps9p and Rabex5 may represent a
novel class of nucleotide exchange factors that modulate the activity
of Vps21p/Rab5.
Stimulating the release of bound nucleotide appears to be the primary
action of guanine nucleotide exchange factors. Based on structural
studies, it has been postulated that guanine nucleotide exchange
factors may interfere with the ability of GTPases to bind
Mg2+ and therefore reduce their affinity for guanine
nucleotides (reviewed in Ref. 53). Upon nucleotide release, GTPases and
nucleotide exchange factors form relatively stable complexes that are
devoid of guanine nucleotide (21, 47-49). The formation of this
complex has been demonstrated in vitro by depleting guanine
nucleotide or by introducing mutations in GTPases that reduce the
affinity for guanine nucleotide (21, 47-49). The strong association
seen between Vps9p and Vps21p-S21N relative to wild-type Vps21p
suggests that Vps9p act in a similar manner. Serine 21 of Vps21p is
located in the highly conserved nucleotide-binding pocket shared by
most GTPases. Where structural information is known, the hydroxyl group of this conserved serine (or threonine) has been shown to participate in coordinating both the GDP-bound Rab proteins are extracted from the target membrane by Rab
GDP dissociation inhibitor, or GDI, and exist as Rab/GDI heterodimers
in the cytoplasm (56, 57). Upon arrival to a specific membrane
compartment, Rab GDI is thought to be displaced by the action of a GDF,
which allows Rab proteins to relocate to the membrane (9, 58, 59).
Subsequently, a nucleotide exchange factor would activate the Rab
protein by stimulating GDP release followed by GTP binding.
Interestingly, all detectable Vps9p is found in a soluble cell fraction
(27). The cytosolic localization of Vps9p indicates that recruitment of
Vps21p to the membrane is independent of, and most likely precedes, the action of Vps9p, which is consistent with the temporal model of Rab
recruitment/activation (58, 59). Since Vps9p appears to be able to
reach any membrane compartment, there must be a mechanism that prevents
Vps9p from activating Vps21p at the target membrane, where most of the
GDP-bound Vps21p is localized (25). It is likely that there is a
specific GDF for Vps21p and that the localization of the GDF determines
where Vps21p is to be activated by Vps9p. In this process, the GDF may
present Vps21p to Vps9p at the correct donor membrane. It is
conceivable that mutations that inactivate the Vps21p GDF would result
in similar phenotypes associated with vps9 and
vps21 mutants as well as other class D vps
mutants (19). We are currently examining the role of proteins
affected in other class D vps mutants in the process of
membrane recruitment and activation of Vps21p.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200 trp1-
901 lys2-801 suc2-
9(17), CBY1 (SEY6210;
vps9
1::HIS3) (27), GTY1 (BHY10 (25);
vps9
2::HIS3 vps21
3::NEO). GTY1 was
generated by transforming CBY20 (27) (BHY10;
vps9
2::HIS3) with a
ClaI/PvuII vps21
3::NEO
fragment of pBHY21-78. Escherichia coli strains used were:
CW2642 (araE201
araFGH::kanr
srl::Tn10 recA59 pro
[F'
proAB+ lacIq ] (31), M15
(Nals Strs
rifs lac
ara
gal
mtl
F
recA+ uvr+) (QIAGEN, Inc.),
XL1Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44) (Stratagene), and MC1066 (F
lacXYZ
hsr
hsm+ spsL galW galK
trpC9830 leuB600 pyrF::Tn5) (32). Bacterial strains were
grown in LB medium containing ampicillin (50 µg/ml) and/or kanamycin
(25 µg/ml) (33). Yeast strains were grown in 2% peptone, 1% yeast
extract, 2% glucose (YPD) or in synthetic medium (SM) supplemented
with the appropriate amino acids as required (34). Polymerases,
restriction and modifying enzymes were purchased from Roche Molecular
Biochemicals, Life Technologies, Inc., or New England Biolabs.
[35S]Pro Mix, [
-32P]GTP,
peroxidase-conjugated anti-rabbit IgG, and peroxidase-conjugated anti-mouse IgG were purchased from Amersham. [3H]GDP was
from NEN Life Science Products Inc. Monoclonal anti-HA antibody was
obtained from Berkeley Antibody Co. Production of antiserum to Vps21p
and carboxypeptidase Y has been described previously (25). Canine
His-Rab5 protein was a gift from Marino Zerial (35).
-Galactosidase filter
assays were performed as described previously (30).
2::HIS3
vps21
3::NEO) were transformed with low or high copy
Vps9HAp expression plasmids (pGT9HA-1 and pGT9HA-2, respectively),
and/or with low or high copy Vps21p expression plasmids (pGBY21-5 and
pBHY21-28, respectively), or a low copy Vps21p-S21N expression plasmid
(pBHY21-11). Strains were grown in appropriate SM to an
OD600 of 0.8 and spheroplasts were generated, lysed, and
treated with the cross-linking agent dithiobis(succinimidylpropionate) as described previously (25). Vps21p and its associated proteins were
isolated by immunoprecipitation as described previously (42). The
immunoprecipitates were resolved in duplicate by SDS-PAGE and
transferred to nitrocellulose membranes. The presence of Vps9HAp and
Vps21p was determined by Western analyses (43) using HA monoclonal
antibodies or Vps21p antiserum and the ECL chemiluminescent detection
system (Amersham, Inc.).
-D-thiogalactopyranoside for 2 h at 37 °C. The cells were harvested and washed twice
with a buffer containing 50 mM Tris-HCl and 100 mM NaCl. The cell pellets were resuspended in lysis buffer
(50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 100 µg/ml phenylmethylsulfonyl fluoride), and lysed using a Bead-Beater (BioSpec Products). The lysate was cleared by
sequential centrifugation and the supernatant was subjected to ammonium
sulfate fractionation. Proteins that precipitated at 40-60%
saturation were collected. The desalted proteins were loaded onto a
DEAE-Sephacel column pre-equilibrated with buffer A (50 mM
Tris-HCl, pH 7.5, 2 mM MgSO4). Proteins that
eluted with buffer A containing 100 mM NaCl were
precipitated by adding ammonium sulfate to 80%. Dialyzed samples
(Buffer A) were loaded onto a Q2 anion exchange column (Bio-Rad)
equilibrated with buffer A. Proteins were eluted with a linear gradient
of 0 to 150 mM NaCl in buffer A. Ypt7p was purified from
E. coli CW2642 carrying pKKYPT7. Induction and purification
was carried out similarly to the Vps21p procedure with minor
modifications. Proteins were eluted from a DEAE-Sephacel column with
buffer A containing 200 mM NaCl. The eluate was
concentrated, dialyzed, and loaded onto a Q2 column. Proteins were
eluted with a linear gradient of 0-300 mM NaCl in buffer A.
-32P]GTP
(6.5 × 104 cpm/µl) for 30 min at 30 °C. The
reactions were stopped by adding 1 ml of ice-cold buffer C (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4) and subsequently filtered through nitrocellulose membranes (0.45-µm pore). The membrane filters were washed twice with 5 ml of ice-cold buffer C and dried. The amount of radioactivity associated with the
filters was determined using a liquid scintillation counter.
-32P]GTP-Vps21p in 250 µl
of 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 15 µM [3H]GDP
(1.5 × 104 cpm/µl), or [
-32P]GTP
(1.3 × 104 cpm/µl) for 30 min at 30 °C. One
hundred µl of the preloaded Vps21p was mixed with an equal volume of
50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 mM MgSO4, 4 mM GDP with or without
Vps9p and incubated at 30 °C. At each time point, 25 µl of the
mixture was removed and diluted in 1 ml of ice-cold buffer C. Protein-bound [3H]GDP or [
-32P]GTP was
determined as in the binding assay described above. The assay was
carried out in the same manner for Ypt7p and His-Rab5. Nucleotide
exchange activity was monitored by incubating Vps21p in the presence or
absence of Vps9p in 200 µl of 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 mM MgSO4, 4 mM GTP, and [
-32P]GTP (100 µCi/ml).
Vps21p-associated [
-32P]GTP was determined as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification and nucleotide binding of
wild-type and Vps21p-S21N. A, wild-type and S21N mutant
Vps21p were purified as described under "Experimental Procedures."
Proteins in the peak fractions from the Q2 column chromatography were
subjected to SDS-PAGE (10%) and stained with Coomassie Brilliant Blue.
Wild-type Vps21p (lane 1) and Vps21p-S21N (lane
2) are indicated by the arrowhead. B,
purified wild-type or Vps21p-S21N (54 pmol each) was incubated with 50 µM [3H]GDP (solid bar) or 50 µM [ -32P]GTP (hatched bar)
for 30 min at 30 °C. The reaction mixture was mixed with ice-cold
binding buffer and filtered through nitrocellulose membranes (0.45-µm
pore) to separate proteins from unbound nucleotides. The amount of
protein-bound nucleotides that remained on the nitrocellulose membranes
was determined by scintillation counting.
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Fig. 2.
Vps21p-S21N interacts with Vps9p in the yeast
two-hybrid system. L40 yeast expressing Gal4AD-Vps9p and
LexA-Vps21p-S21N, Gal4AD-Vps9p and LexA-Vps21p wild-type, pGADGH
(Gal4AD vector) and LexA-Vps21p-S21N, pGADGH and LexA-Vps21p wild-type,
and Gal4AD-Vps9p and pVJL11 (LexA vector) were streaked onto
A, a YNB plate lacking tryptophan, leucine, and histidine
and onto B, a YNB plate lacking tryptophan and leucine and
incubated at 30 °C for 72 h. These yeast strains were also
patched onto a YNB plate lacking tryptophan and leucine and grown for
72 h at 30 °C. C, the patches were then transferred
to a nitrocellulose membranes, the transferred cells were lysed, and
subjected to a colorimetric -galactosidase assay.
-galactosidase was determined by an activity assay (see
"Experimental Procedures"). Only cells coexpressing the
LexA-Vps21p-S21N fusion and the Gal4AD-Vps9p fusion had observable
-galactosidase activity (patch 1, Fig. 2C). Neither the
LexA-Vps21p (wild-type) fusion together with the Gal4AD-Vps9p fusion
nor the LexA-Vps21p or Gal4AD-Vps9p fusions alone expressed the
-galactosidase reporter (patches 2-5, Fig. 2C). These
results were completely consistent with those generated using the
HIS3 reporter gene, indicating that the Vps9p may have a
preferential binding affinity for a GDP-bound form of Vps21p.
3, vps9
2) expressing various
combinations of Vps9HAp, Vps21p, and Vps21p-S21N from low or high copy
number plasmids, were lysed and the lysates were treated with the
homobifunctional cross-linking agent,
dithiobis(succinimidylpropionate), or left untreated. The lysates were
then subjected to immunoprecipitation with Vps21p antiserum and the
immunoprecipitates were resolved by SDS-PAGE. The resolved
immunoprecipitates were subjected to Western analysis, using Vps21p
antiserum to detect Vps21p or HA monoclonal antibodies to detect
Vps9HAp. When extracts generated from strains coexpressing Vps9HAp and
Vps21p-S21N from CEN-based (low copy number) vectors were
treated with cross-linking agent, two proteins with masses of
approximately 64 and 65 kDa were detected in the immunoprecipitates
(Fig. 3A, lane 4). These
proteins correspond to two forms of Vps9HAp that have increased
relative masses due to the covalent addition of cross-linker molecules.
Importantly, Vps9HAp was not present in immunoprecipitates when
cross-linking agent was omitted (Fig. 3A, lane 5), when
Vps9HAp was not expressed (Fig. 3A, lanes 2 and
3), or when Vps21p or Vps21p-S21N was not expressed (Fig.
3A, lane 1). In addition, approximately 10-fold more Vps9HAp
was cross-linked to Vps21p-S21N when Vps9HAp was expressed from a
multicopy vector (2 µm) (Fig. 3A, lane 6). A weak
interaction between Vps9HAp and wild-type Vps21p was also uncovered.
When extracts generated from a strain overexpressing both Vps9HAp and
wild-type Vps21p were treated with cross-linking agent, a small but
significant amount of Vps9HAp was detected in the immunoprecipitates
(Fig. 3A, lane 8) that was not seen in the absence of
cross-linking agent (Fig. 3A, lane 9).
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Fig. 3.
Vps21p-S21N is more efficiently cross-linked
to Vps9HAp than wild-type Vps21p. Protein lysates were generated
from GTY1 (vps9 2 vps21
3) spheroplasts carrying the
following plasmids in the combinations denoted above panel A
and panel B: pGT9HA-1 (CEN VPS9HA), pGT9HA-2
(2-µm VPS9HA), pBHY21-28 (2-µm VPS21),
pBHY21-21 (2-µm VPS21-S21N), and pBHY21-11 (CEN
VPS21-S21N). Each lysate was divided into two aliquots. One was
treated with the cross-linking agent, dithiobis(succinimidylpropionate)
(400 µg/ml), and the other was left untreated. Each aliquot was then
subjected to immunoprecipitation with Vps21p antiserum and duplicate
samples of the resulting immunoprecipitates were resolved by SDS-PAGE.
The resolved proteins were transferred to nitrocellulose membranes and
probed with: A, HA antibodies to visualize Vps9HAp; and
B, Vps21p antiserum to visualize the Vps21p levels in each
strain. In panel B, the film was exposed to the ECL
reagent-treated membranes for 3 s (B, lanes
1-3), 10 s (B, lanes 4-7), and 1 s (B, lanes 8 and 9).
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Fig. 4.
Overproduction of Vps9p enhances the guanine
nucleotide exchange activity of Vps21p. Vps21p (88 pmol) was
preloaded with 15 µM [3H]GDP and then
diluted in 2 mM unlabeled GDP in the presence of lysis
buffer ( ), 13,000 × g supernatant fractions
(approximately 440 µg of protein) from SEY6210 (
) or SEY6210
carrying pPS92 (
) in a 200-µl assay mixture and incubated at
30 °C. At each time point, equal aliquots were removed, mixed with
ice-cold buffer, and filtered through nitrocellulose membranes. The
membrane filters were washed with buffer, dried, and counted.
Radioactive GDP that remained protein bound is presented as a function
of time.
2) and the ability of His-tagged Vps9p to complement
vps9
2 mutant phenotypes was analyzed. Cells that lack
functional Vps9p missort and secrete soluble vacuolar proteins (27). In
the case of the vacuolar hydrolase carboxypeptidase Y (CPY),
vps9 mutants secrete the Golgi-modified precursor form of
the enzyme (p2CPY), whereas wild-type cells properly localize p2CPY
from the Golgi to the vacuole where it is processed to its mature
active form (mCPY) (27). In the experiment shown in Fig.
5A, wild-type, vps9
2, and vps9
2 cells expressing
(His)6-Vps9p were pulse-labeled with
[35S]methionine and cysteine. Cell lysates were
generated, CPY was immunoprecipitated from each lysate and the
immunoprecipitates were resolved by SDS-PAGE. In wild-type cells, newly
synthesized CPY was delivered to the vacuole as evidenced by the
presence of the mature vacuolar form of the enzyme (mCPY) (Fig.
5A, lane 1). In the vps9
2 cells CPY delivery
to the vacuole was blocked and Golgi-modified p2CPY accumulated (Fig.
5A, lane 2). When recombinant (His)6-Vps9p was
expressed in the vps9
2 cells, CPY was processed to its
mature form (mCPY) (Fig. 5A, lane 3) indicating that
vacuolar protein transport was restored. This result demonstrates that the amino-terminal hexahistidine tag does not interfere with the function of Vps9p.
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Fig. 5.
Characterization of recombinant
(His)6Vps9p. A, SEY6210 (wild-type,
lane 1), CBY20 (vps9 2, lane 2), and
CBY20 carrying pEMBLVPS9 (vps9
2 + (His)6Vps9p, lane 3) were labeled with
[35S]methionine and cysteine for 10 min at 30 °C.
Unlabeled methionine and cysteine were added and the cells were
incubated for another 30 min. Cells were lysed and subjected to
immunoprecipitation with CPY antiserum. Immunoprecipitated CPY was
resolved by SDS-PAGE and visualized by fluorography. B,
(His)6-Vps9p was purified as described under
"Experimental Procedures." The peak fraction from Q2 column
chromatography was subjected to SDS-PAGE (10%) and stained with
Coomassie Brilliant Blue. The 58-kDa (His)6-Vps9p is
indicated by an arrowhead.
). Stimulation of Vps21p-dependent GTP association was
also observed (Fig. 6B). This result clearly demonstrates that Vps9p stimulates the guanine nucleotide exchange of Vps21p. Interestingly, isoprenylation of Rab3 proteins was shown to be necessary for the action of the Rab3 guanine nucleotide exchange factor
(Rab3 GEP) (13). However, isoprenylation of Vps21p is not required for
Vps9p-dependent nucleotide exchange.
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Fig. 6.
Vps9p stimulated both GDP displacement and
GDP/GTP exchange. A, Vps21p (100 pmol) was preloaded
with 15 µM [3H]GDP and diluted in 2 mM unlabeled GDP in the absence ( ) or presence of 25 (
), 50 (
), or 100 (
) pmol of purified (His)6-Vps9p
in a 200-µl assay mixture. Protein-bound [3H]GDP was
determined as described in the legend to Fig. 4. B,
nucleotide exchange activity was monitored by incubating Vps21p (200 pmol) in the presence (
) or absence (
) of Vps9p (200 pmol) in 200 µl of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM MgSO4, 4 mM GTP, and
[
-32P]GTP (100 µCi/ml). Vps21p-associated
[
-32P]GTP was determined as described in the legend to
Fig. 4.
-32P]GTP in the presence or
absence of Vps9p. As shown in Fig.
7A, pre-bound
[3H]GDP release was stimulated by adding purified Vps9p.
However, the same amount of Vps9p did not stimulate the release of
[
-32P]GTP from Vps21p (Fig. 7B). This
result demonstrates that Vps9p only stimulates GDP release but not GTP
release from Vps21p and that Vps9p does not stimulate the GTPase
activity of Vps21p.
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Fig. 7.
Vps9p does not stimulate GTP/GDP
exchange. A, Vps21p (177 pmol) was preloaded with 15 µM [3H]GDP or B, 15 µM [ -32P]GTP and diluted in 2 mM unlabeled GDP in the absence (
) or presence (
) of
(His)6-Vps9p (88 pmol). Protein-bound [3H]GDP
and [
-32P]GTP were determined as described in the
legend to Fig. 4.
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Fig. 8.
Nucleotide exchange activity of Vps9p is
specific for Vps21p and Rab5. A, purified Vps21p (490 pmol); B, Rab5 (490 pmol); C, Ypt7p (490 pmol)
were preloaded with 15 µM [3H]GDP and
diluted in 2 mM unlabeled GDP in the absence ( ) or
presence (
) of (His)6-Vps9p (180 pmol). Protein-bound
[3H]GDP was determined as described in the legend to Fig.
4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant strains (25). Second,
purified Vps9p possesses specific guanine nucleotide exchange activity
for Vps21p/Rab5. Vps9p did not stimulate nucleotide exchange of yeast
Ypt7p, which is another Rab involved in later stages of vacuolar
protein transport. Consistent with the inability of Vps9p to stimulate
nucleotide exchange of Ypt7p, mutations in the YPT7 gene
manifest several phenotypes distinct from vps21 or
vps9 mutants. Finally, vps9 mutants show no
obvious defects in other protein localization pathways. Based on these
data, we conclude that the major role of Vps9p in vacuolar protein
transport is to stimulate the guanine nucleotide exchange of Vps21p.
-phosphate of GTP and Mg2+
(54). Therefore, loss of the hydroxyl group in the serine 21 to
asparagine Vps21p mutant is anticipated to result in a protein with low
affinity for GTP, which could stabilize the GDP-bound Vps21p-exchange
factor complex and/or the nucleotide-free Vps21p-exchange factor
complex. An interesting distinction of Vps21p/Vps9p association is that
Vps9p does not seem to associate with GTP-bound Vps21p. Association
between many GTPases and their nucleotide exchange factors occurs
irrespective of the bound guanine nucleotide. This subsequently results
in GTP as well as GDP release, at least in vitro (48, 49,
55). In our assays, nucleotide exchange of GTP-bound Vps21p was not
stimulated by Vps9p, indicating that Vps9p has very low, if any,
affinity for GTP-bound Vps21p. The strong preference of Vps9p for
GDP-bound Vps21p would drive the equilibrium in only one direction
generating the GTP-bound, activated form of Vps21p in
vivo.
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ACKNOWLEDGEMENTS |
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We thank members of the Horazdovsky laboratory for helpful discussions during the course of this study. We also thank Michael White for yeast two-hybrid system reagents and suggestions and Marino Zerial for providing us with purified Rab5.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-55301 (to B. F. H.) and March of Dimes Foundation Grant 5-FY97-0119 (to B. F. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Biology, Utah State University, Logan,
UT 84322-5305.
§ To whom correspondence should be addressed. Tel.: 214-648-3148; Fax: 214-648-9156; E-mail: bhoraz{at}biochem.swmed.edu.
2 H. Hama and B. F. Horazdovsky, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: GDI, GDP dissociation inhibitor; GDF, GDI dissociation factor; GEF, guanine nucleotide exchange factor; PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; vps, vacuolar protein sorting; CPY, carboxypeptidase Y.
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REFERENCES |
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