From the Department of Cell and Developmental Biology and The Institute for Human Gene Therapy, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160
Received for publication, September 27, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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Rab GTPases are localized to distinct subsets of
organelles within the cell, where they regulate SNARE-mediated membrane
trafficking between organelles. One factor required for Rab
localization and function is Rab GDP dissociation inhibitor (GDI),
which is proposed to recycle Rab after vesicle fusion by extracting Rab
from the membrane and loading Rab onto newly formed transport
intermediates. GDI is composed of two domains; Rab binding is mediated
by Domain I, and the function of Domain II is not known. In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19
gene, was targeted in a genetic screen to obtain mutants that
might lend insight into the function of this domain. In one
gdi1 mutant, the cytosolic pools of all Rabs tested were
depleted, and Rab accumulated on membranes, suggesting that this mutant
Gdi1 protein has a general defect in extraction of Rab from membranes.
In a second gdi1 mutant, the endosomal/vacuolar Rabs
Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but
localization of Ypt1p and Sec4p were not significantly affected. Using
an in vitro assay which reconstitutes Gdi1p-mediated
membrane loading of Rab, this mutant Gdi1p was found to be defective in
loading of Vps21p but not Ypt1p. Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that
membrane-associated Rab may regulate recruitment of GDI-Rab from the
cytosol, possibly by regulating a GDI-Rab receptor. We conclude that
Domain II of Gdi1p is essential for Rab loading and Rab extraction, and
confirm that each of these activities is required for Gdi1p function
in vivo.
Monomeric GTPases of the Ras superfamily regulate myriad cellular
pathways, and the study of the mechanisms by which they confer
regulation, and the mechanisms by which they are themselves regulated,
are central problems in cell biology. Members of the Rab subfamily
regulate trafficking of macromolecules between organelles by directly
regulating the machinery responsible for vesicle-mediated trafficking.
Each unique inter-organelle transport pathway within the cell is
regulated by a distinct Rab GTPase, and this is reflected in the
localization of each Rab to distinct organelles (1, 2). The enzymatic
cycle of several Rabs is well characterized, and they work in much the
same way as Ras. Guanine nucleotide exchange factors activate
Rab by facilitating exchange of GDP for GTP, activating downstream
signaling by specific recruitment of effector proteins which bind
activated Rab. GTPase activating proteins terminate Rab signaling by
accelerating hydrolysis of GTP (1). Of the Rab guanine nucleotide
exchange factors characterized to date, each is specific for a single
Rab, but the characterized Rab GTPase activating proteins exhibit
overlapping specificities in vitro (3-7).
Membrane association of Rab is mediated primarily by geranylgeranyl
moieties covalently attached to C-terminal cysteine residues (8). In
addition, however, a soluble pool of Rab also exists, associated with
Rab GDP dissociation inhibitor
(GDI),1 a protein first
identified by its ability to inhibit activation of Rab (9). The
importance of GDI function is reflected in the finding that mutations
in the gene encoding the human GDI result in mental retardation (10).
Rab GDI extracts inactive (i.e. GDP-bound conformation) Rab
from membranes in what is thought to constitute the initial step of Rab
recycling. In the second step of Rab recycling, Rab is loaded onto
membranes. Little is known about the mechanism by which GDI-Rab
complexes are targeted to membranes. Presumably, only a subset of
intracellular membranes can serve as the target for GDI-mediated Rab
loading, although this idea has not been rigorously tested. Accessory
factors that regulate GDI function have been postulated to exist,
although none have been identified. An activity that stimulates
dissociation of Rab9 from GDI, but does not catalyze nucleotide
exchange, has been termed GDI displacement factor and suggested to
facilitate loading of Rab9 onto endosomal membranes (11). Hypothetical factors that facilitate Rab membrane extraction by GDI have been termed
Rab recycling factors (12).
The crystal structure of the In the study presented here, we have addressed several key issues
regarding the mechanism by which GDI-Rab complexes are targeted to
membranes using genetic and biochemical approaches with the budding
yeast, Saccharomyces cerevisiae. Yeast contains a single, essential gene encoding Rab GDI (GDI1/SEC19) which has been
shown to interact with multiple Rabs by a variety of assays (16). We
have focused our studies on two Rabs that function in the vacuolar protein sorting pathways, Vps21p, required for biosynthetic transport between the Golgi and the late endosome (17, 18), and for early
endosome-to-late endosome transport (19), and Ypt7p, a Rab required for
late endosome-to-vacuole transport (20, 21) and for homotypic fusion of
vacuoles (22). Regarding Gdi1p, we have focused our efforts on
investigating the function of the helical domain, as this domain has
been implicated in membrane targeting.
Strains and Media--
S. cerevisiae strains used for
these studies are listed in Table I.
Yeast strains were grown in standard yeast extract, peptone, dextrose
(YPD) (23), yeast extract, peptone, fructose (YPF), or synthetic media
(SM) with essential amino acid supplements (23) as required for
maintenance of plasmids. Standard bacterial media (24) was used for
Escherichia coli cultures. Transformation of S. cerevisiae strains was done by the lithium acetate method of Ito
et al. (25) with single-stranded DNA employed as carrier (26). E. coli transformations were done according to the
method of Hanahan (27).
DNA Methods--
Standard DNA manipulations (28) were carried
out with restriction endonucleases, DNA modification enzymes, and
Taq polymerase from Promega, Roche Biochemicals, or New
England Biolabs. DNA was prepared using Qiagen prep column and DNA
fragments were gel-purified using a Qiagen gel extraction kit.
The plasmid vector used for random mutagenesis of GDI1,
pPG3, was constructed by digesting pRS414 with SalI, then
trimming the ends with T4 DNA polymerase, and reclosing the plasmid.
Next, the wild-type GDI gene (12) was cloned into the
SpeI and ClaI sites. Versions of mutant Gdi1p
tagged with the hemagglutinin (HA) epitope were generated by replacing
the NcoI to NotI fragment of pPGgdi11 or pPGgdi29
with the NcoI to NotI fragment from wild-type, HA-tagged GDI1 (12). The membrane trafficking and Rab
fractionation phenotypes of HA-tagged gdi1 mutant cells was
indistinguishable from the phenotypes of native gdi1 mutants
(data not shown).
Mutagenesis of GDI1--
Gapped plasmid repair mutagenesis was
used to restrict mutagenesis to codons encoding amino acids 81 to 175 of GDI1 (29). To prepare the vector for transformation,
~50 µg of pPG3 was digested with NcoI and
SalI, and the gapped vector was gel-purified. For polymerase
chain reaction amplification of GDI1, four separate PCRs
were performed. The standard concentrations for each nucleotide in the
mutagenic PCRs was 200 µM. Four PCRs were done, and in each PCR the concentration of one of the nucleotides was dropped as
follows: dATP, 0.2 µM; dGTP, 0.13 µM; dCTP,
0.13 µM; dTTP, 0.2 µM. These concentrations
were determined empirically to produce approximately equal yields of
amplified product, and product from all four PCRs was pooled. For
transformation, gapped plasmid and PCR-mutagenized DNA were transformed
into CBY71. We used plasmid shuffling on 5-fluororotic acid plates to
exchange the wild-type GDI1 allele on a URA3
vector for the gap-repaired allele (30). Two screens were done to
identify mutants affected in GDI1 function. To screen for
temperature-conditional growth, transformation plates were
replica-plated to two YPD plates and one set of plates was maintained
at room temperature and the other set was incubated at 37 °C for 3 days. Colonies were identified which grew well at room temperature, but
not at 37 °C. To screen for gdi1 mutants defective in
vacuolar protein sorting, transformation plates were replica-plated to
two YP fructose plates and allowed to grow at room temperature
overnight. The next morning, one set of plates was transferred to
37 °C for 3 h, and then both plates were screened for secretion
of CPY-Invertase using a previously described colorimetric plate assay
(31). Colonies were identified that secreted the CPY-Invertase fusion
protein at both temperatures, or predominantly at 37 °C.
Metabolic Labeling and Protein Sorting Assays--
Yeast
cultures were radiolabeled using previously published procedures (17,
32). Immunoprecipitations of CPY were done using the method of Klionsky
et al. (33).
Subcellular Fractionation--
Subcellular fractionation
experiments were carried out as described (34). For experiments
requiring temperature shift, cells were converted to spheroplasts, and
then resuspended (5 A600/ml) in SM media
containing 1 M sorbitol. Each culture was split in half,
and one-half was incubated with shaking at 26 °C while the other was
placed at 37 °C as indicated for 30 min before harvesting.
GDI Functional Assays--
Coimmunoprecipitations of Rabs by
HA-tagged Gdi1p were done as described in Luan et al. (12).
Antisera used for immunoblotting various Rabs have been described (12,
17).
For in vitro GDI assays, acceptor membranes were prepared as
follows. Cells (relevant genotype indicated in the legends to the
figures) were converted to spheroplasts, and lysed on ice in reaction
buffer (RB) (20 mM HEPES, pH 7.5, 200 mM
sorbitol, 50 mM potassium acetate, 1 mM
dithiothreitol, 1 mM EDTA, 5 mM magnesium
chloride, and protease inhibitors (mini-complete, Roche Molecular
Biochemicals)) at a concentration of 20 A600/ml
with 20 strokes of a glass tissue homogenizer. Cell extracts were
cleared by centrifugation at 300 × g for 5 min. The
extract was then loaded onto a 1-ml cushion of sucrose (60% in RB),
and centrifuged at 100,000 × g for 1 h in a
Sorvall S80 AT3 rotor. The buffer-sucrose interface was collected in a
minimal volume, and the protein concentration was determined. The donor
fractions were prepared from cells expressing wild-type or mutant
gdi1 alleles. Cleared cell extracts were prepared as
described above. After clearing, the extracts were centrifuged at
100,000 × g for 1 h in a Sorvall S80 AT3 rotor,
and the supernatant fraction was collected and the protein
concentration determined.
Standard assays contained 80 µg of membrane protein in the acceptor
fraction, 100 µg of S100 protein in the donor fraction, and GDP or
GTP
To test the effect of trypsin digestion on acceptor membranes, ~500
µg of membranes (protein concentration), harvested using the standard
protocol, were incubated with 90 µg of trypsin (Life Technologies) at
37 °C for 30 min. Protease inhibitor (AEBSF) (Calbiochem) was then
added to 10 mM, and the membranes were harvested by
centrifugation at 100,000 × g for 1 h.
Mock-treated membranes were prepared exactly as above, except trypsin
was omitted. The membrane pellets were resuspended in RB containing 1 mM AEBSF and protein concentrations were determined.
Standard loading assays were set up using equivalent volumes of mock
treated and trypsin-treated membranes.
For Rab loading saturation experiments (Fig. 6B), standard
GDI assays were set up using only 20 µg of membrane protein, and different amounts of cytosol prepared from wild-type cells. Each reaction contained 100 µM GTP Mutagenesis of Gdi1p Helical Domain--
Previous
structure/function studies of mammalian and yeast GDI have identified
the apex of Domain I as the region which binds Rab, and this region has
been termed the Rab-binding platform (Fig.
1) (12-15). GDI also contains a highly
conserved Domain II composed primarily of
Each of the mutants obtained was characterized by pulse-chase analysis
of newly synthesized carboxypeptidase Y (CPY). Biogenesis of CPY can be
informative for identifying trafficking defects in multiple segments of
the secretory and vacuolar protein sorting pathways due to
compartment-specific modifications (addition of N-linked
carbohydrate and proteolytic processing) which can be easily monitored
by SDS-polyacrylamide gel electrophoresis. Based on this analysis, the
collection of mutants could be categorized into two classes. Class I
mutants (2 isolates) exhibited a rapid, temperature-conditional block
in endoplasmic reticulum to Golgi transport similar to the original
gdi1/sec19-1 mutant (Fig. 2), and did not secrete any CPY from the cell at restrictive temperature (data not shown). Class II mutants (28 isolates) accumulated
Golgi-modified p2CPY, especially when assayed at 26 °C (Fig. 2), and
a small amount of p2CPY (~5%) was secreted into the media (data not
shown). Analysis of protein secretion in representative mutants from
each class revealed a strong block in secretion from the Class I mutant (gdi1-11) cells, while secretion from the Class II mutant
(gdi1-29) was similar to wild-type cells (Fig. 2). DNA
sequencing of three mutants (2 Class I mutants and 1 Class II mutant)
revealed that each had multiple mutations, but interestingly, all three
mutants had changes in the same two codons encoding Met140
and Gly141 (Fig. 1).
Gdi1p-mediated Rab Recycling Is Affected in gdi1 Mutants--
It
is thought that the primary function of Rab GDI is to recycle inactive
Rab GTPases after vesicle fusion back to an appropriate donor membrane
(e.g. a transport vesicle). Two biochemical activities of
GDI contribute to this function, extraction of inactive Rab-GDP from
the membrane and loading of Rab-GDP onto membrane (Fig. 1). To
determine which activities of Rab GDI are affected in the mutants, we
examined the distribution of multiple Rabs in wild-type cells and in
the gdi1-11 and gdi1-29 mutants at permissive
(26 °C) and restrictive temperatures (37 °C) (Fig.
3). Mutant cells were incubated at 26 or
37 °C for 30 min prior to cell fractionation, and the relative
amounts of Rab in pellet fractions containing membranes and soluble
cytosolic fractions were determined by immunoblotting with antibodies
to Ypt1p (endoplasmic reticulum to Golgi transport), Vps21p (Golgi to
endosome, and early endosome to late endosome transport), Ypt7p (late
endosome to vacuole transport and vacuole-vacuole fusion), and Sec4p
(Golgi to plasma membrane transport). In wild-type cells, the majority
of each Rab fractionated with membranes in the 100,000 × g pellet, in agreement with previously published studies. In
the Class I gdi1-11 mutant, the distribution of all four
Rabs was affected compared with wild-type cells. When cells were
preincubated at restrictive temperature (37 °C), the cytosolic fractions of each Rab were nearly completely depleted, with a concomitant increase in membrane-associated pools. Compared with wild-type cells, little change in Rab fractionation was observed when
gdi1-11 cells were preincubated at permissive temperature (26 °C), suggesting that this Gdi1p mutant protein has a general, temperature conditional defect in membrane extraction of Rab.
In the gdi1-29 mutant, Vps21p and Ypt7p accumulated in the
cytosol when cells were preincubated at 26 or 37 °C (Fig.
3A). Localization of Ypt1p (Fig. 3B) or Sec4p
(data not shown) were not significantly affected. In three independent
experiments, an average of ~60% of Vps21p, and ~90% of Ypt7p were
detected in the cytosolic fractions of mutant cells compared with ~20
and 10%, respectively, in wild-type cells. Thus, in the
gdi1-29 mutant, the membrane-associated pools of the
endosomal/vacuolar Rabs, Vps21p and Ypt7p, were depleted, with a
concomitant increase in the cytosolic pools of these Rabs. These
results suggest that the gdi1-29 mutant Gdi1 protein is
defective preferentially in membrane loading of Vps21p and Ypt7p.
Mutant Gdi1 proteins unable to bind Rab due to mutations in the
Rab-binding platform are not functional and do not support the
essential function of GDI1 in vivo (12). Thus, it was
important in the case of the gdi1-29 mutant to determine
whether Rab accumulated in the cytosol bound to Gdi1p. To test this, we
introduced HA epitope-tagged versions of wild-type and mutant Gdi1p
that allowed us to immunoprecipitate Gdi1p under native conditions
(12). Spheroplasts were incubated at 26 or 37 °C for 30 min, then
lysed and membranes were removed by centrifugation. Gdi1p was
immunoprecipitated from the soluble fraction under native conditions
and the immunoprecipitated material was then probed with antibodies to
Vps21p, Ypt7p (Fig. 3), Ypt1p, and Sec4p (data not shown). All four Rab
proteins coimmunoprecipitated with wild-type and gdi1-29
mutant Gdi1 proteins, indicating that the gdi1-29 mutant
Gdi1 protein is not defective in binding Vps21p or Ypt7p.
Mutant Gdi1 Proteins Are Defective in Rab Extraction and
Loading--
Accumulation of Rab in the membrane fraction, with a
concomitant loss in the cytosol fraction, suggests that the
gdi1-11 mutant protein is defective in membrane extraction
of Rab, whereas accumulation of Rab in the cytosol bound to Gdi1p
suggests that the gdi1-29 mutant protein is defective in Rab
loading. To test these hypotheses, an in vitro assay was
developed that monitored Rab loading and extraction. For these
experiments, we focused on Vps21p because localization of this Rab in
the gdi1 mutants was representative of the other three Rabs
assayed in the experiments described above, and because several factors
which regulate, or are regulated by, Vps21p are known. In the first
experiment, membranes were collected from wild-type cells and served as
"acceptor" membranes. Cytosol from wild-type cells, cleared of
membranes by centrifugation, was used as a source of Gdi1p-Vps21p
complexes (the "donor" fraction). The two fractions were mixed,
incubated at 30 °C for 30 min with or without added GDP or GTP
We next compared Vps21p loading and extraction activities of extracts
prepared from gdi1 mutant strains. When cytosolic extracts prepared from gdi1-11 cells incubated at permissive
temperature (26 °C) were used as the source of Gdi1p-Vps21p
complexes, Vps21p was recovered in the membrane fractions, even in the
absence of added GTP
When cytosolic extracts prepared from the gdi1-29 mutant
were tested, very little change in Vps21p distribution was observed under any conditions, notably, even in the presence of GTP Membrane Loading of Rab Requires Membrane-associated
Proteins--
It is generally regarded that GDI-mediated membrane
loading of Rabs onto distinct organelles involves membrane-associated proteins which serve as receptors to recruit GDI-Rab complexes from the
cytosol, although none have been identified yet (35). We tested the
hypothesis that membrane-associated proteins are required for
Gdi1p-mediated loading of Vps21p by treating acceptor membranes with a
protease to destroy associated proteins, re-isolating the membranes,
and using them in a loading assay. If membrane-associated proteins are
required for Rab loading, protease treatment should destroy Rab
acceptor activity of membranes. Substantially less Vps21p was loaded
onto trypsin-treated membranes compared with mock-treated membranes
(Fig. 6A), indicating that Rab
acceptor activity was severely, although not completely, compromised by trypsin treatment. Another prediction of the receptor-mediated model is
that loading of Vps21p should be saturable. To test this, loading
reactions were set up using a small amount of membranes prepared from
wild-type cells and increasing amounts of cytosol. The results (Fig.
6B) indicate that loading of Vps21p is saturable, as has
been reported for several other Rabs (36-39).
A putative GDI receptor is expected to bind Gdi1p, Rab, or both. We
adapted the in vitro GDI assay to test for a possible role
of membrane-associated Rab on GDI-mediated Rab loading. To do so, we
took advantage of the fact that VPS21 is not an essential gene, so we were able to prepare membranes from a vps21
We next examined loading by the gdi1-29 Gdi1p mutant, using
vps21 Gdi1p Domain II Required for Rab Loading and Extraction--
We
have used genetic and biochemical approaches to gain insight into the
function of Gdi1p Domain II, and the mechanisms by which Gdi1p-Rab
complexes are targeted to different organelles. A region of
GDI1 encoding Domain II and a small portion of Domain I was
targeted for random mutagenesis in a genetic screen to identify loss-of-function mutations in this region. Analysis of these mutants clearly indicates that this domain is important for both Gdi1p-mediated Rab loading and extraction in vivo and in vitro.
At least two different models of Domain II function can explain the
results. One possibility is that Domain II binds directly to
membrane-localized accessory proteins required for loading or
extraction. In this view, the gdi1-29 loading-defective
mutant protein is predicted to not interact properly with a putative
receptor, and the gdi1-11 mutant protein does not interact
sufficiently with a factor required for extraction. A second
possibility is that the conformation of Gdi1p changes upon binding Rab
and that this conformational change is required to extract and load
Rab. According to this model, the conformation of the
gdi1-29 mutant would favor stable binding of Rab, and the
structure of the gdi1-11 mutant protein would favor the Rab
empty conformation (at restrictive temperature). This is the most
likely explanation for the gdi1-11 mutant, as all Rab
accumulates on membranes, yet the gdi1-11 mutant protein is
predominantly cytosolic (data not shown). If this interpretation of the
gdi1-11 phenotype is correct, then the extraction defect of
the gdi1-11 mutant protein is a consequence of an inability to bind Rab under restrictive conditions. It is important to note that
the extraction-defective mutant protein encoded by the
gdi1-11 allele is stable under restrictive conditions (data
not shown), suggesting the protein does not globally unfold releasing
Rab nonspecifically.
Gdi1p-mediated Rab Loading--
Membrane-associated receptors
which recruit Gdi1p-Rab complexes from the cytosol have been postulated
to exist, however, no functional receptor for GDI-mediated loading of
any Rab has been identified (35). Evidence supporting this hypothesis
includes saturation loading of Rab (Fig. 6), and that protease
treatment of membranes inhibits Rab loading activity (Fig. 6) (36-39).
A candidate receptor for GDI-mediated Rab loading in mammalian cells is
prenylated Rab acceptor protein, Pra1, a predicted membrane protein
which was first identified in a two-hybrid screen with Rab3, and
subsequently demonstrated to bind many other (but not all) Rabs (40,
41). Pra1 also has been shown to bind weakly to GDI and inhibit
in vitro extraction of Rab3a by recombinant GDI (42). The
yeast protein Yip3p is homologous to Pra1, and was identified in a
two-hybrid screen using the yeast Rab Ypt31p, indicating that it can
bind Rab, but little else is known about Yip3p
function.2 Another
Rab-binding membrane protein identified in the same screen was Yip1p,
which is essential for secretion (43). We have found that
overexpression of YIP1 or YIP3 does not suppress
Rab mislocalization in the gdi1-29 loading-defective mutant
(data not shown). Nonetheless, the sequence and functional similarities
between human Pra1 and yeast Yip1p and Yip3p suggest that Yip1p and
Yip3p are likely candidates for regulating Rab and possibly Gdi1p
functions in yeast.
An important implication of the analysis of the gdi1-29
loading-defective mutant is that Gdi1p-dependent
localization of Vps21p and Ypt7p appear to be coregulated. This
conclusion is based on the observations that localization of Vps21p and
Ypt7p (but not Ypt1p or Sec4p) were affected in the gdi1-29
mutant. One possible explanation for these results is that the rates at
which different Rabs are recycled by GDI are not equivalent, so that in
gdi1 mutants Rab localization is perturbed in nonequivalent
ways. If, for example, Vps21p and Ypt7p are rapidly recycled compared
with Ypt1p and Sec4p, then loss of Gdi1p-mediated loading would affect
localization of Vps21p and Ypt7p more significantly than Ypt1p or
Sec4p. An alternative explanation is that loading-defective Gdi1p fails to interact sufficiently with a membrane receptor that functions for
only a subset of Rabs including Vps21p and Ypt7p. The target organelles
for Vps21p and Ypt7p loading have not been identified, and this will be
crucial information for distinguishing these possibilities.
Membrane-associated Vps21p had a dramatic effect on in vitro
loading of Vps21p; in vitro membrane loading of Vps21p by
the gdi1-29 mutant Gdi1p mutant was defective, but only when
membranes containing Vps21p were used in the assays. Thus, the presence of Vps21p on membranes more faithfully reconstitutes the Gdi1p-Rab loading reaction. Because membranes lacking Vps21p were still active
for Vps21p loading, we speculate that the activity of an as yet
unidentified Gdi1p-Vps21p receptor may be regulated by direct
association of Vps21p with the putative receptor. Under conditions
where receptor is not bound to Rab (i.e. vps21 Membrane Extraction of Rab by GDI--
Little is known about
regulation of GDI-mediated extraction of Rab. Only Rab in the inactive,
GDP-bound state is a ligand for GDI (44), so activation of Rab by
GDP/GTP exchange, and hydrolysis of GTP, are presumed to be key factors
regulating the size of cytosolic Rab pools. In this context, it is
surprising that the amount of Vps21p in the cytosol of wild-type and
vps9
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-isoform of bovine GDI has been solved
to high resolution (13, 14). The molecule consists of two structural
domains: Domain I contains all of the
strands present in the
molecule, and a smaller domain, Domain II, is composed primarily of
-helix (13). Previous mutagenesis studies have clearly shown that
Rab binds Domain I in a region termed the Rab-binding platform (12, 13,
15). The function of helical Domain II is not known, although it has
been postulated to interact with protein receptors important for
membrane targeting of GDI-Rab complexes (12-15). Domain II of the
highly related Mrs6 protein, a component of the Rab prenylation
machinery, has been implicated in binding enzymatic subunits of the
prenylation machinery, consistent with the role of Domain II in
proteins of the CHM/GDI family in recognition of other factors required
for Rab function. Recently, a highly mobile loop in GDI, termed the
"GDI effector loop," was identified in a region linking Domain I
and Domain II, and this loop was also suggested to constitute an
important element of the membrane targeting region of GDI (14).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S. cerevisiae strains used in this study
S as indicated. The final assay volume was 300 µl, and standard
assay conditions were 30 °C for 30 min. After the assay, 1 ml of RB
was added to each reaction and mixed. Diluted assay mixtures were
centrifuged for 1 h at 100,000 × g in a Sorvall S45A rotor. Supernatant fractions were collected, precipitated with
trichloroacetic acid, washed with cold acetone twice, and then
dried. Sample buffer (2.5 mM Tris, pH 6.8, 12% SDS, 6 M urea, 5%
-mercaptoethanol, 10% glycerol) was then
added to each pellet. An equivalent amount of sample buffer was added
to each pellet fraction. Rabs were visualized by immunoblotting and
quantitated by densitometry using NIH Image software (developed at the
U.S. National Institutes of Health and available on the Internet).
S. Vps21p was visualized
using an enhanced chemifluoresence kit (Pierce) and the signals were
quantitated on a STORM PhosphorImaging system (Molecular Dynamics), or
by densitometry of ECL films.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix, and the function
of this domain is not known. Using a PCR-based random mutagenesis
procedure, we targeted amino acids 54-225 of yeast Gdi1p, which
includes nearly all of Domain II (amino acids 128-228), to obtain new
mutants that might provide insight into the function of this conserved domain. A small portion of Domain I was also targeted due to restraints imposed by the location of restriction enzyme sites. After a plasmid shuffle scheme to swap wild-type and mutagenized GDI1 genes,
we screened for temperature-conditional loss-of-function mutants by
identifying colonies which grew at 23 °C, but grew poorly or not at
all at 37 °C, and in a second screen of the same transformants, we
identified mutants which missorted vacuolar proteins at 23 °C and/or
at 37 °C using a colorimetric-based plate assay (31). Two mutants
were obtained which grew well at 23 °C, but extremely slowly at
37 °C. Twenty-eight mutants were obtained which missorted vacuolar
proteins at 23, 37 °C, or at both temperatures.
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Fig. 1.
Structure and function of Rab GDI.
A, the Rab GTPase cycle is illustrated. GEF, Rab
guanine nucleotide exchange factor; GAP, Rab GTPase
activating protein. B, a ribbon diagram of bovine GDI-
(13) is shown with several features highlighted. The region of yeast
GDI1 mutagenized in this study is highlighted in dark
gray, and the previously defined Rab-binding platform is contained
in the dashed box. Both the Rab loading-defective
(gdi1-29) and the Rab extraction-defective
(gdi1-11) mutant Gdi1 proteins contained two mutations in
common, M140I and G141S, contained in the rectangle. The
Rab loading-defective mutant contained two additional mutations,
Q55R and T105A, which are boxed. The Rab
extraction-defective mutant contained four additional mutations, S116P,
I155S, V179A, and R211G, which are circled.
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Fig. 2.
Protein transport in gdi1
mutants. A, CPY sorting assays. The indicated
cultures were incubated at 26 or 37 °C for 15 min prior to labeling
for 10 min with Easy-Tag. Chase solution was then added and the cells
were incubated for 30 min. Cultures (media and cells) were then
harvested by trichloroacetic acid precipitation and processed
for immunoprecipitation of CPY. B, secretion assay. The
indicated cultures were labeled and chased as described above. After
the chase period, cells were removed from the culture by
centrifugation, and proteins in the media fraction were collected by
trichloroacetic acid precipitation and visualized by
autoradiography.
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Fig. 3.
Distribution of Rab GTPases in
gdi1 mutants. A, Vps21 and Ypt7p.
Spheroplasts of the indicated cultures were preincubated at 37 °C
for 30 min prior to cell lysis. After lysis, cell extracts were
centrifuged (100,000 × g) for 1 h to separate
membranes from soluble components, and equivalent amounts of pellet
fractions containing cell membranes, and supernatant fractions
containing soluble components, were separated by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and immunoblotted with
antibodies to the indicated Rab. Only the 37 °C data are shown and
similar results were obtained with cells incubated at 26 °C. To
determine whether Rabs accumulate in the cytosol bound to Gdi1p, 25 A600 equivalents of cells expressing HA
epitope-tagged wild-type or gdi1-29 mutant Gdi1p were
preincubated at 37 °C for 30 min, cells were lysed and membranes
removed by centrifugation. Gdi1p was immunoprecipitated under native
conditions with antibodies to the HA epitope, and immunoprecipitated
material was then probed with antisera to the indicated Rab.
B, Ypt1p. Cells were preincubated at 26 or 37 °C for 30 min and then fractionated as described above and Ypt1p was visualized
by immunoblotting. WT, wild-type.
S,
then diluted with ice-cold reaction buffer. Membranes were then
recovered by centrifugation and the amounts of Vps21p in the pellet and
supernatant fractions, containing membranes and cytosol, respectively,
were determined by immunoblotting (Fig.
4). Comparison of the amounts of Vps21p
in the starting donor and acceptor fractions, versus
the amounts in the membrane and cytosol fractions after incubation
together revealed whether net loading or extraction had occurred during
the incubation. When cytosol from wild-type cells was used, there was
little change in the distribution of Vps21p between membranes and
cytosol in the absence of added nucleotide, or in the presence of GDP,
indicating that little loading or extraction of Rab occurred under
these conditions. An alternative interpretation of this result is that Rab loading and extraction are balanced. When GTP
S was included in
the reaction, Vps21p was recovered predominantly in the membrane fraction, indicating that Vps21p had been loaded onto membranes under
these conditions. Rab loading required physiological temperature, as
little Vps21p net loading was observed when the reaction was incubated
on ice (Fig. 5). Immunoblotting of
membrane fractions with antibodies to Gdi1p revealed that Gdi1p was not
detected in the membrane fraction, even in reactions kept on ice (data not shown), indicating that recovery of Vps21p in the membrane fraction
was not the result of nonspecific association of Gdi1p-Vps21p complexes
with membranes. Importantly, cytosolic Vps21p coimmunoprecipitated with
antibodies to Gdi1p in the presence of 150 µM GTP
S
(data not shown). This result suggests that the requirement for GTP
S in the assay is not simply to dissociate Rab from Gdi1p in the cytosol,
but rather, it may be required to prevent extraction of membrane-loaded
Rab.
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Fig. 4.
A, Rab loading by the gdi1-11
mutant Gdi1 protein is independent of added nucleotide. Membranes
purified from wild-type cells were used as acceptor membranes, and
cytosol prepared from wild-type cells (top row) or
gdi1-11 mutant cells grown at 26 °C (bottom
row) was used as a source of donor Gdi1p-Rab complexes. Components
were mixed on ice without added nucleotide (No NT), with 100 µM GDP or 100 µM GTP S, then incubated at
30 °C for 30 min. One reaction was kept on ice for the entire
incubation period ("ICE"). At the end of the incubation,
the reaction was diluted with 1 ml of ice-cold reaction buffer, and
membranes were separated from soluble material by centrifugation. The
amount of Vps21p in the pellet (P) fractions containing
membranes, and the supernatant fractions (S) was determined
by immunoblotting. The amount of Rab in the membrane and cytosol
fractions before mixing is shown in the "Input" lanes.
No NT, no added nucleotide.
View larger version (48K):
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Fig. 5.
The gdi1-29 mutant Gdi1p is
defective in membrane loading of Rab. Standard Gdi1p functional
assays were carried out using membranes prepared from wild-type cells,
and cytosol from wild-type (top row) or gdi1-29
cells grown at 26 °C (bottom row). The same fractions
were also probed with antibodies to Ypt1p.
S, the presence of 100 µM GDP, or
when no nucleotide was added (Fig. 4). Little Vps21p was loaded in a
reaction kept on ice. These results are consistent with the
interpretation that the gdi1-11 mutant protein can load Rab
but is defective in membrane extraction of Rab, and they also indicate
that the gdi1-11 mutant protein is defective in the in
vitro GDI assay, even under conditions which are permissive
in vivo.
S (Fig. 5). Surprisingly, similar results were obtained when Ypt1p was monitored, despite the observation that localization of Ypt1p is not
affected significantly in vivo. Taken together, these
results confirm that Rab loading in this assay is mediated by Gdi1p,
and that the mutant Gdi1 proteins are defective in membrane extraction (gdi1-11) and loading (gdi1-29) of Rab.
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Fig. 6.
Gdi1p-mediated membrane loading of Vps21p
requires membrane-associated proteins and is saturable.
A, protease inhibits Rab acceptor activity of membranes.
Membranes purified from vps21 cells were treated with
trypsin, then reisolated by centrifugation. Control membranes were mock
treated. Membranes were used in a standard loading assay with 100 µM GTP
S. B, Gdi1p-mediated loading of
Vps21p is saturable. Standard in vitro loading assays were
carried out using 20 µg of membranes (protein equivalent) from
wild-type cells, and increasing amounts of cytosol from wild-type cells
as indicated. Components were mixed on ice and GTP
S was added to 100 µM. The amount of Vps21p in each fraction was quantitated
by densitometry and is plotted below.
null strain, and test them for acceptor activity, using cytosol
prepared from wild-type cells. Vps21p loading required substantially
more GTP
S compared with standard assays using membranes from
wild-type cells which contain Vps21p (Fig.
7). Over three independent experiments, ~60% of Vps21p was loaded in the presence of 150 µM
GTP
S, while greater than 90% of Vps21p was loaded under standard
assay conditions (100 µM GTP
S) using membranes
prepared from wild-type cells containing Vps21p. These results suggest
that membrane-associated Vps21p strongly influences Gdi1p-mediated
loading of cytosolic Vps21p.
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Fig. 7.
Membrane-associated Vps21p influences
Gdi1p-mediated membrane loading. To monitor only in
vitro Rab loading, acceptor membranes were prepared from
vps21 cells (which lack this Rab) and used in a standard
GDI assay. Loading of Vps21p required more GTP
S compared with assays
employing membrane from wild-type cells containing Vps21p (compare with
Fig. 5). In the bottom panels, results from identical assays
using gdi1-29 donor cytosol prepared from cells incubated at
26 °C are shown. Vps21p was loaded onto membranes lacking Vps21p, in
contrast to results obtained when membranes containing Vps21p were used
(compare with Fig. 5).
membranes and cytosol prepared from
gdi1-29 cells (Fig. 7). Surprisingly, when cytosol prepared
from gdi1-29 cells was tested, no significant difference
between wild-type and gdi1-29 loading of Vps21p was
observed. These results indicate that membrane-associated Vps21p is
required to recapitulate in vitro the Rab loading defect of
the gdi1-29 mutant protein. Because loading of cytosolic,
Gdi1p-associated Vps21p clearly does not require membrane-associated
Vps21p, these results suggest that the activity of an as yet
unidentified membrane-associated factor, possibly a Gdi1p-Vps21p
receptor, is regulated by membrane-associated Vps21p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
membranes), Gdi1p-mediated loading is facilitated, however, when
receptor is limiting, due to association with Rab, differences in the
abilities of wild-type and mutant Gdi1p to load Rab manifest. This
mechanism could provide a means to regulate Rab loading according to
the amount of Rab engaged with membrane-associated proteins such as downstream effectors, which would in turn affect the amount of Rab
available to bind receptor. An alternative possibility is that gross
changes in the membrane composition of acceptor membranes prepared from
vps21
cells facilitated loading by the gdi1-29 loading-defective Gdi1 protein. This possibility is unlikely because membranes prepared from other deletion strains which lack endosomal components that interact with Vps21p (Pep12p t-SNARE and Vac1p effector) behave like wild-type membranes in in vitro
loading assays.3 Membranes
from these cells are expected to have gross changes in composition
similar to vps21
cells.
(encoding a guanine nucleotide exchange factors for
Vps21p (5)) cells is not significantly different compared with
wild-type cells.4 Other
factors must regulate extraction of Rab by Gdi1p, and these putative
factors have been termed Rab recycling factors (12). Observations
supporting this idea include the finding that expression of Gdi1p
mutant proteins unable to bind Rab still associate with membranes in a
protease-sensitive manner (12), and that expression of high levels of
Rab binding-defective Gdi1p results in depletion of cytosolic Rab
pools. The collection of new gdi1 mutants described here
should be useful for identifying these and other factors that regulate
Gdi1p function.
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ACKNOWLEDGEMENT |
---|
We thank Mickey Marks for critical reading of the manuscript, and Mickey Marks, Mark Lemmon, Scott Emr, Bill Balch, Gerry Waters, Alisa Kabcenell, Pat Brennwald, and Erfei Bi for helpful discussions and reagents.
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FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed. Tel.: 215-573-5158;
Fax: 215-898-9240; E-mail: cburd@mail.med.upenn.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008845200
2 D. Gallwitz, personal communication.
3 P. M. Gilbert and C. G. Burd, unpublished data.
4 P. M. Gilbert and C. G. Burd, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
GDI, GDP
dissociation inhibitor;
HA, hemagglutinin;
PCR, polymerase chain
reaction;
CPY, carboxypeptidase Y;
AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride;
GTPS, guanosine
5'-3-O-(thio)triphosphate.
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