From the Departments of Cell and Molecular
Biology-IMM 11, The Scripps Research Institute, La Jolla, California
92037 and the ¶ Division of Cellular and Molecular Medicine, The
Howard Hughes Medical Institute, University of California, San Diego,
School of Medicine, La Jolla, California 92093-0668
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ABSTRACT |
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Guanine nucleotide dissociation inhibitor (GDI)
is an essential protein required for the recycling of Rab GTPases
mediating the targeting and fusion of vesicles in the exocytic and
endocytic pathways. Using site-directed mutagenesis of yeast
GDI1, we demonstrate that amino acid residues required for
Rab recognition in vitro are critical for function in
vivo in Saccharomyces cerevisiae. Analysis of the
effects of Rab-binding mutants on function in vivo reveals
that only a small pool of recycling Rab protein is essential for
growth, and that the rates of recycling of distinct Rabs are
differentially sensitive to GDI. Furthermore, we find that membrane
association of Gdi1p is Rab-independent. Mutant Gdi1 proteins unable to
bind Rabs were able to associate with cellular membranes as efficiently
as wild-type Gdi1p, yet caused a striking loss of the endogenous
cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when
expressed at levels of the normal, endogenous pool. These results
demonstrate a potential role for a new recycling factor in the
retrieval of Rab-GDP from membranes, and illustrate the importance of
multiple effectors in regulating GDI function in Rab delivery and
retrieval from membranes.
Rab proteins are membrane-associated small GTP-binding proteins
which regulate the targeting and fusion of vesicle carriers transporting cargo between compartments of the exocytic and endocytic pathways of both yeast and mammalian cells (1). Following vesicle fusion, Rab proteins in the GDP-bound form are recycled by members of
the guanine nucleotide dissociation inhibitor
(GDI)1 gene family (reviewed
in Ref. 2 and 3). The We have solved the structure of bovine The critical role of GDI family members in the regulation of membrane
transport throughout the endocytic and exocytic pathways stresses the
importance of understanding the contribution of individual amino acid
residues in directing interaction of GDI not only with Rab, but
importantly, upstream or downstream effectors involved in Rab
recycling. The fact that the yeast S. cerevisiae contains only a single copy of GDI1 which is essential for cell
growth (5) provides an excellent tool to extend our previous studies on
GDI function in ER to Golgi transport in vitro using
mammalian cells (12). To date, only one additional factor, a guanine
nucleotide displacement factor has been proposed to function in the
GDI-Rab cycle, in this case facilitating the delivery of Rab to
membranes by interaction with the GDI-Rab cytosolic pool (14-16).
We now report evidence for a new factor involved in GDI function
in vivo. We find that single point mutations in yeast
GDI1 codons encoding homologous residues of Strains, Media, and Microbiological Methods--
Yeast strains
were grown in standard yeast extract, peptone, dextrose (YPD) (17),
yeast extract, peptone, fructose (YPF), or synthetic media (SM)
supplemented with 2% casamino acids and essential amino acid
supplements (17) as required for maintenance of plasmids.
Transformation of Saccharomyces cerevisiae strains was done
by the lithium acetate method (18) with single-stranded DNA employed as
carrier (19). Standard bacterial media was used for Escherichia
coli cultures. E. coli transformations were done as
described (20).
S. cerevisiae strains used for these studies are as follows.
A gdi1 DNA Methods--
Standard DNA manipulations (21) were used with
restriction endonucleases and modification enzymes from Roche Molecular
Biochemicals, New England Biolabs, or U. S. Biochemical Corp. The
gdi1 Metabolic Labeling, Immunoprecipitations, and Subcellular
Fractionations--
Yeast cultures were radiolabeled using previously
published procedures (23, 24). Strains were labeled with FM4-64
(Molecular Probes, Eugene, OR) as described (25). Immunoprecipitations of CPY were done as described (26). Native immunoprecipitations of HA
epitope-tagged Gdi1p proteins were done with a monoclonal antibody
which recognizes the HA epitope (Roche Molecular Biochemicals). Cells
were lysed (15 OD600 unit per 1 ml) in standard lysis
buffer (20 mM HEPES (pH 6.2), 200 mM sorbitol,
100 mM potassium acetate, 2 mM magnesium
chloride, 1 mM dithiothreitol) and subjected to centrifugation(s) as described under "Results." Anti-HA antibody was added (5 µl per ml of extract), incubated with rocking at 4 °C
for 4 h, and immunoprecipitated with Gamma Bind G-Sepharose (Pharmacia).
For subcellular fractionation studies, spheroplasts made from cells
were labeled and processed as described in Ref. 27. After clearing
extracts of unbroken cells, lysates were centrifuged at 100,000 × g for 1 h to yield P100 particulate (membrane) and S100
soluble (cytosolic) fractions. Fractions were precipitated with
trichloroacetic acid and then processed for immunoprecipitation with
polyclonal rabbit antisera raised against various yeast Rab proteins.
Site-directed Mutagenesis of Yeast GDI1--
To map amino acid
residues of yeast Gdi1p that are important for function, a plasmid
shuffle-based complementation assay (17) was developed that allowed
rapid screening of site-directed point mutants for the ability to
complement a gdi1
We have previously shown that mutation of some of the highly conserved
residues in SCRs 1B and 3B found at the apex of GDI affect the ability
of the mutant GDIs to bind Rab3A in vitro (10) (Fig. 1).
However, the physiological relevance of these observations remained to
be determined. Two other SCRs (2 and 3A) contain conserved residues
that are found on one face of GDI located beneath the Rab-binding
region (10) (Fig. 1). Using the crystallographic structure and the
aligned sequences of all GDIs and REPs comprising the GDI superfamily
as a guide, point mutations changing conserved, surface-exposed amino
acids of the various SCRs that were likely candidate residues for
interactions with Rab or other molecules were constructed. Each was
tested in the complementation assay. In addition, mutations were
introduced to assess other structural features of Gdi1p. These included
the GXG motif found at the base of the Rab-binding region
that is potentially involved in the binding of a putative nucleotide
cofactor. We also examined the importance of the carboxyl-terminal
region containing helix N that supports the putative Rab-binding
platform found at the apex of Gdi1p (10) (Fig. 1).
The results of our initial analysis is shown in Table I. Despite the
extensive collection of point mutants tested, no single amino acid
change was found which resulted in a complete loss of Gdi1p function
other than point mutations predicted to disrupt structure (Table I,
single asterisk). Although we did not observe complete loss of cell
growth for the single point mutants, one mutation, R248A (Table I,
double asterisks), clearly affected Gdi1p function as evident by the
slower growth of this strain. This particular residue was previously
implicated as one of the most important amino acids required for Rab
binding in vitro, reducing binding approximately 60-fold
(10). Because single point mutations of the homologous conserved
residues in yeast Gdi1p did not result in lethal gdi1
phenotypes, it is apparent that even the reduced level of Rab3A binding
measured in vitro for equivalent mammalian GDI mutants (10)
is sufficient to maintain adequate levels of Rab recycling in
vivo for cell survival. These results now establish a working
relationship between in vitro binding assays (that provide
relative measure of GDI-Rab interaction) and the ability of GDI to
recognize Rab under physiological conditions in vivo. Given
the observation that reduced binding is not lethal, we suggest that the
recycling activity of wild-type GDI is likely to be in functional
excess in living cells.
Because the R248A mutant resulted in a modest growth defect, this
mutation was used as a starting point to construct a set of double
mutants. Additional amino acid substitutions were made in either the
same SCR containing R248 (SCR 3B) or in different SCRs, and each of
these was tested in combination with R248A in the complementation assay
(Table II). We also generated double mutants within and between SCRs that did not include the
Arg248 residue to test whether multiple mutations in
regions of Gdi1p flanking the putative Rab-binding platform would yield
defective function. Three double mutant combinations did not complement the gdi1 The R248A,Y44V and R248A,E241S Double Mutants Cause a Growth
Defect in sec19-1 Cells--
To further define the function of the
gdi1 mutant genes, we examined whether selected mutants
could complement the growth defect of sec19-1 cells. The
function of the protein encoded by sec19-1 is
temperature-sensitive resulting from a stop codon near the
carboxyl-terminal end of helix N, leading to the production of a
truncated protein whose folding is thought to be unstable at slightly
elevated (semi-permissive) temperatures (30 °C). Complete loss of
function is observed at 37 °C (28). Consistent with this
interpretation, further truncation resulting in the partial or complete
removal of helix N leads to complete loss of function (data not shown).
Each of the gdi1 mutant genes on CEN vectors was
transformed into a sec19-1 strain (CBY47) and transformants
were selected and maintained at 26 °C. Transformants were then
restreaked and shifted to 37 °C for 3 days and the growth of each of
the strains was assessed. Consistent with the above results, all single
gdi1 mutations suppressed the ts growth defect
(Fig. 2A). In contrast, each
of the double mutations that did not complement the gdi1 Mutants Are Deficient in Rab Binding--
Mutant gdi1
alleles that failed to complement the gdi1
The double mutant genes and their corresponding single mutant genes
cloned into the HA-vector were used to transform a sec19-1 strain. Immunoblotting of total cell lysates derived from these strains
with an HA-specific antibody revealed that a single band of
approximately 52 kDa was present in the R248A,Y44A and R248A,E241S double mutants (Fig. 3A). All
other mutant proteins tested, with one exception (see below Fig.
4A), were stable when
expressed in vivo (Fig. 3A and data not
shown).
To determine if the stable double mutant Gdi1 proteins would bind Rabs,
we used a native co-immunoprecipitation assay to examine their
interaction with three different Rab GTPases that function at distinct
steps of the secretory pathway: Ypt1p, required for ER/Golgi transport
(29); Vps21p, required for Golgi/endosome transport (24, 30); and Ypt7,
which functions in late endosome to vacuole trafficking and
vacuole-vacuole fusion (31, 32). Extracts were prepared from strains
grown at permissive temperature (26 °C), centrifuged at 100,000 × g for 1 h to yield P100 particulate fractions
containing most intracellular membranes, and S100 cytosolic fractions.
HA-Gdi1 proteins were immunoprecipitated from the S100 fractions under
native conditions and the immunoprecipitates were probed with
antibodies against Ypt1p, Ypt7p, and Vps21p.
While each of the Rabs was efficiently co-immunoprecipitated with
wild-type HA-Gdi1p, none were co-immunoprecipitated with the double
mutant HA-Gdi1 proteins (Fig. 3B). Notably, significant but
reduced and variable amounts of each of these Rabs were co-precipitated by each of the corresponding single mutants (Fig. 3B), a
result consistent with the observation each of these single mutants
affect binding of Rab3A in vitro (10), yet still support
cell growth. Significantly, the differential effect of various point
mutants on the recovery of Rab raises the possibility that residues in GDI may play specific roles in the recognition of separate Rab species.
Identical results were obtained when we inactivated the endogenous
sec19-1 mutant Gdi1p by temperature shift to the restrictive temperature (37 °C) for 30 min before cell lysis, indicating that the lack of Rab binding observed was not simply a consequence of
competition by the endogenous temperature-conditional Gdi1 protein
(data not shown).
A concern in the above experiments was that the immunoprecipitation
conditions may not accurately reflect binding of Gdi1p to Rab in
vivo due to dilution effects following cell homogenization and
subsequent washing steps during immunoprecipitation. To address this
concern, we first examined whether the membrane association of
wild-type and double HA-tagged mutants was the same or different when
membranes prepared these cell lines were washed in an identical fashion
to that used for co-immunoprecipitation with Rabs. This condition would
mimic the potential effects on dilution of the endogenous concentration
of HA-Gdi1p and Rabs following washing. As shown in Fig. 3C,
under these conditions, both wild-type and HA-tagged double mutants
showed identical levels of binding to membranes. The experiments
suggest that the failure to bind Rabs represents at least one critical
defect responsible for the growth phenotype of single and double Gdi1p mutants.
Mutation of the Rab-binding Region Does Not Affect Association of
Mutant Gdi1p with Membranes--
To determine if any of the mutant
Gdi1 proteins were affected in their ability to partition between the
cytosol and intracellular membranes, fractions from the experiment
described above were probed with the anti-HA antibody using
immunoblotting (Fig. 4A). Intriguingly, we found that while
approximately 30% of wild-type Gdi1p was found associated with the
P100 fraction, a nearly identical level was observed for the GDI
mutants. A negligible signal was detected in the vector only control.
Interestingly, the mutant containing substitutions at both
Arg248 and the GXG motif (R248A,G16A,G18A) could
not be detected. One possibility is that the expression of the protein
from this vector is somehow inhibited, even though at least 15 plasmids
that differed by only single point mutants expressed stable protein
(Fig. 4A and data not shown). Alternatively, we suggest that
the inability to detect protein suggests that the conserved
GXG motif found at the base of the Rab-binding platform (10)
is critical for the folding and/or stability of the protein.
To further address the ability of mutant GDI to recognize membranes in
response to loss of Rab recognition in vivo, a cell homogenate was prepared from sec19-1 cells containing the
vector only. Either the whole cell homogenate (Fig. 4B, a-c)
or a P100 fraction containing membranes (Fig. 4B, e-f) were
incubated for 30 min at 30 °C with cytosol prepared in an identical
fashion from cells expressing HA-tagged wild-type GDI and HA-tagged
241/248 double mutant. Following incubation, P100 membrane fractions
were isolated from each sample, washed, and the amount of HA-tag bound to membranes was determined by immunoblotting. As shown in Fig. 4B, the levels of binding of wild-type and the double mutant
were identical even in the presence of cytosolic Sec19-1 Gdi1p (Fig. 4B, compare lane b and c (cytosolic
Sec19-1 Gdi1p present) to e and f (cytosolic
Sec19-1 Gdi1p absent)). Moreover, binding was dependent on a
membrane-associated receptor. As shown in Fig. 4C,
pretreatment of membranes with trypsin prevented binding of wild-type
and mutant HA-Gdi1p. These in vitro results are consistent with the conclusion that mutant GDI that cannot recognize Rab in
vivo, still retains the ability to bind efficiently to membranes. Moreover, binding is weakly dominant over Sec19-1 at the permissive temperature and completely dominant at the semipermissive temperature given the growth inhibitory phenotype (Fig. 2).
Intracellular Membrane Trafficking in gdi1 Mutants--
Gdi1p is
required for numerous membrane trafficking steps that involve
potentially 11 different Rab proteins in yeast (33). We therefore
examined our collection of mutants for secretory transport function.
Given the ability of most gdi1 single mutants to support
growth, it was possible that there were selective defects in Rab
function given the differential effects of various single mutants on
Rab binding using the co-immunoprecipitation assay (Fig.
3B). We monitored Gdi1p function in the early secretory pathway (ER/Golgi) and the vacuolar protein sorting/endocytic (VPS)
pathway by examining the processing of carboxypeptidase Y (CPY), and
secretion by monitoring the release of HSP150 into the medium. In
pulse-chase immunoprecipitation assays, the conversion of the ER p1CPY
precursor form (67 kDa) to the Golgi-modified p2CPY (69 kDa) precursor
reflects transport through the ER and Golgi, and conversion of p2CPY to
mature CPY (mCPY, 61 kDa) in the vacuole reflects transport through the
vacuolar protein sorting pathway. CPY biosynthesis was assayed at 10- and 30-min chase points which allowed us to visualize each of these CPY
biosynthetic transport steps.
For the viable Gdi1p mutants, pulse-chase assays were conducted with
strains in which the mutant gdi1 alleles were the sole source of Gdi1p. Of those tested, the Y44V, E241S, and R248A single mutants exhibited defects in CPY biosynthesis which reflected partial
kinetic delays in transport2 (see below) consistent with
reduced binding of Rab (Fig. 3B). However, in each case,
nearly all of the CPY was properly processed by late time points for
each mutant, a result consistent with the cell growth
phenotypes.2
To test the inviable gdi1 double mutants, plasmids encoding
them and the corresponding single mutations were transformed into a
sec19-1 strain and CPY biosynthesis was assayed. Cultures
were incubated at permissive (26 °C) or restrictive temperature
(37 °C) for 10 min prior to labeling, then labeled for 10 min, and chased for 10 or 30 min. At the permissive temperature, in all strains,
only p2CPY was present at the 10-min chase point, and by 30 min, all
p2CPY was converted to mCPY. At the restrictive temperature (37 °C),
a tight and rapid block in ER to Golgi transport was evident in the
sec19-1 strain as an accumulation of p1CPY at both early and
late chase points (Fig. 5A, upper
left panel), and this defect was complemented by the wild-type
GDI1 plasmid.3 In
sec19-1 cells transformed with single gdi1 mutant
plasmids, we again observed significant kinetic defects in CPY
biosynthesis, but only at the restrictive temperature, a result
identical to those observed using the gdi1
In contrast to the effects of single point mutations, CPY biosynthesis
was severely affected at 37 °C in sec19-1 strains
expressing each of the double gdi1 mutant proteins
(R248A,Y44V and R248A,E241S). In the R248A,Y44V strain, p1CPY
accumulated, indicating a potent ER to Golgi transport block with only
a very small amount (<5%) of mCPY present at the 30-min chase point
(Fig. 5A, bottom left panel). The transport block observed
in the R248A,E241S strain was not as severe, as we observed partial
complementation of the transport block imposed by the
sec19-1 mutation. In both cases, no further processing of
CPY was observed beyond the level seen after 30 min (Fig.
5A, data not shown). The fact these mutants fail to support
growth (Table II) is consistent with the interpretation that they can
only support an exceedingly limited level of recycling of a critical Rab(s).
Because the analysis of CPY biosynthesis in sec19-1
R248A,Y44V cells did not reveal any significant transport defects in
the early secretory pathways at 26 °C despite the partial growth
defects at the permissive temperature and complete block at the
semipermissive condition (Fig. 2), we also tested for possible late
secretory defects (Golgi to cell surface) by assaying secretion of
Hsp150p from mutant cells at 30 °C (semipermissive condition).
Hsp150p is a 341-amino acid O-glycosylated protein which is
rapidly secreted into the media from wild-type cells (34). Mutant
strains were pulse labeled for 5 min, chased for various amounts of
time, and Hsp150 was immunoprecipitated from extracts derived from
cells or from the media fraction. If a late step in transport is
blocked by the R248A,Y44A mutant, we should expect to see a marked
depression in secretion despite the fact that HSP150 is an extremely
rapidly transported protein, largely appearing in the medium during the initial pulse period (Fig. 5B). As shown in Fig.
5B, we did not detect a decreased rate of secretion of
HSP150 in response to the presence of the R248A,Y44V and R2148A, E241S
double mutants. All of the newly synthesized Hsp150p was rapidly
secreted from cells by the end of the labeling period (Fig.
5B). Thus, there were no detectable differences in secretion
from these mutant strains at 30 °C, implying an important function
for GDI in an as yet untested Rab-dependent pathway other
than the secretory pathway affecting cell growth.
Endocytic Membrane Transport Is Impaired in gdi1 Mutant
Cells--
To expand our understanding of the Rab-binding region to
Rab-regulated events involved in internalization from the cell surface, we examined the effects of the Rab-binding deficient double mutants on
the endocytic pathway by following the uptake and transport of the
lipophilic dye FM4-63. When added to growing cells, FM4-64 intercalates
into the plasma membrane and is transported to the vacuolar membrane in
an energy-, time-, and temperature-dependent manner. Thus,
it can be used to monitor endocytosis of bulk membrane (35). Wild-type
and sec19-1 cells were incubated at either the permissive
temperature or shifted to restrictive temperature (37 °C) for 10 min, then FM4-64 was added to the growth media, and the cultures were
incubated for 10 min to allow labeling of the plasma membrane. Fresh,
prewarmed media was then added to initiate a chase period during which
aliquots of cells were removed at 10- and 45-min chase points and the
FM4-64 dye was visualized by fluorescence microscopy (Fig.
6).
The uptake of FM4-64 to the vacuole of wild-type and sec19-1
cells was indistinguishable at the permissive temperature (data not
shown). In sec19-1 cells carrying a complementing
GDI1 plasmid, most of the FM4-64 had been delivered to the
vacuolar membrane at the 10-min chase point at 37 °C, although a
small amount still persisted in a few puncta distributed throughout the
cytoplasm (Fig. 6, top left panels). By 45 min of chase,
nearly all of the FM4-64 was in the vacuolar membrane. In contrast,
sec19-1 cells at the 10-min chase point contained numerous
cytoplasmic puncta, with little FM4-64 being delivered to the vacuole.
At the 45-min time point, the vacuolar membrane became labeled,
although cytoplasmic puncta remained along with large vesicular
structures clustered together near the vacuole (Fig. 6, top right
panels). Thus, the sec19-1 mutation slows, but does not
completely block the endocytic transport of FM4-64 to the vacuole.
When FM4-64 uptake was observed in sec19-1 cells expressing
either of the double mutant proteins (R248A,Y44V or R248A,E241S), trafficking of FM4-64 to the vacuole was impaired much more severely than in sec19-1 cells alone (Fig. 6, bottom
panels). At the 10-min chase point at 37 °C, the dye was
distributed throughout the cytoplasm in a very diffuse pattern
indicating the accumulation of a multitude of small structures, in
contrast to the relatively few cytoplasmic puncta present in
sec19-1 cells. By the 45-min chase point, the dye was
observed in vacuolar membranes, although numerous large non-vacuolar
vesicles were also present, consistent with the phenotype of the
sec19-1 cells at this time point. These results suggest that
the prominent effects of sec19-1 and Gdi1p double mutants were on rapid kinetic events occurring during early steps in the transit of dye through endocytic compartments.
Electron microscopy was used in an effort to visualize membrane
trafficking intermediates that might accumulate in gdi1
mutants. Strains were prepared for analysis by growing at the
permissive temperature (26 °C), then each culture was divided and
one-half was transferred to 37 °C and incubated for 1 h while
the other half remained at 26 °C. Cells were then fixed and prepared
for electron microscopy. Fig. 7
(panels A and B) shows that sec19-1 R248A,Y44V cells and sec19-1 cells grown at the permissive
temperature were similar in appearance. This morphology is
characteristic of wild-type cells (25).
In contrast to the above results, numerous aberrant but unidentified
membrane structures were observed in the sec19-1 strain at
the restrictive temperature. Surprisingly, we did not observe the
striking accumulation of vesicles that were observed following slow
depletion of Gdi1p (5). Moreover, at the restrictive temperature, there
were prominent differences between the sec19-1 strain and sec19-1 R248A,Y44V strains (Fig. 7, panels C and
D). Particularly evident in the latter was the appearance of
numerous 200-250 nM diameter vesicular structures
throughout the cytoplasm of double mutant cells (Fig. 7, panel
E). In addition to the accumulation of large vesicles, the
vacuoles of sec19-1 R248A,Y44V cells often appeared
fragmented and irregularly shaped. In the most striking cross-sections,
large electron-lucent bodies appeared to be clustered along the
vacuolar membrane, giving the vacuolar membrane a punched in appearance
(Fig. 7, panel C, arrow). The EM analyses are consistent with the numerous puncta observed during endocytosis of FM4-64. These
results suggest that a severe defect in vesicular traffic leads to the
accumulation of distinct transport intermediates as a consequence of
the more dominant effect of the double mutants. This defect appears to
be distinct from that of the sec19-1 temperature-sensitive defect.
Rabs Are Loaded onto Membranes by Gdi1p at Different Rates--
In
yeast, in vivo depletion of wild-type Gdi1p over a 15-h time
period by shut-off of GDI1 expression under control of an inducible promoter, results in the loss of the cytosolic pool of Sec4p
(a Rab present on post-Golgi secretory vesicles) and concomitant
accumulation of Sec4p on membranes (5). While these results demonstrate
the importance of recycling of Rab from membranes, they do not address
whether Gdi1p is, in addition, required for membrane loading of Rab as
shut-off would be expected to remove both the Rab-bound and free pools
over the 15-h time period.
To address the above question, we used the sec19-1 strain to
rapidly inactivate the endogenous Gdi1p by shift to the restrictive temperature and monitored the distribution of several Rabs by subcellular fractionation. In order to rigorously quantitate any change
in distribution that might occur, rather than use immunoblotting, strains were labeled at the permissive temperature (26 °C) for 15 min, and then chased for 30 min to allow equilibration of the nascent
radiolabeled Rab pools with the total intracellular Rab pools. The
cultures were then split and one-half of each was transferred to the
restrictive temperature (37 °C) while the other was maintained at
the permissive temperature. After 30 min, cells were lysed, membrane
(P100) and cytosol (S100) fractions were generated by centrifugation,
and Ypt1p, Ypt7p, and Vps21p were immunoprecipitated from the membrane
and cytosol fractions. During this short time period the endogenous
sec19-1p pool is stable, but is completely inactive as is apparent by
the block in transport of CPY (Fig. 5, panel A).
Inactivation of Sec19-1p by shift to the restrictive temperature and
incubation for 30 min led to only a partial shift of the cytosolic
pools of Ypt1p and Vps21p to the membrane (Fig. 8, (top panel) Ypt1p and
(bottom panel) Vps21P, lanes a and b). In contrast, the entire cytosolic pool of Ypt7p was shifted to the
membrane fraction within 30 min of transfer to 37 °C (Fig. 8,
middle panel, lanes a and b). Note
that the time of exposure of the gels for cytosolic Rabs were 3-fold
longer than that of the membrane-bound pool, suggesting that the major
pool of these Rab proteins is membrane-bound. These results are
quantitated in Fig. 9. The strikingly
distinct effects of rapid inactivation of Sec19-1 Gdi1p on the
redistribution of individual Rab species may reflect differences in
rates of retrieval of a Rab protein at a particular step of the
secretory pathway (see "Discussion"). Moreover, the ability to
detect a cytosolic Ypt1p and Vps21p after 30 min at 37 °C suggests
that the primary defect in Sec19-1p is not due to a misfolding event
occurring at the restrictive temperature following delivery of Rab to
membranes. Rather, that the function of the cytosolic Sec19-1p·Rab
complex is sensitive to the temperature-induced folding defect. Thus,
GDI is essential for Rab loading of membranes.
Double Mutants Reveal the Requirement for a Novel Factor in Rab
Recycling--
A GDF has been proposed to be required for Rab9
delivery from the cytosolic pool complexed to GDI (14-16). The
requirement for other factors, including a possible accessory recycling
factor involved in extraction of Rab-GDP following vesicle fusion have not been implicated to date. To test for this possibility, we examined
the affect of expressing gdi1 double mutants unable to bind
Rabs on the membrane/cytosol distributions of Ypt1p, Ypt7p, and Vps21p
using the protocol outlined above for the distribution of Rabs in the
sec19-1 strain at the permissive and restrictive temperatures.
In the presence of either the R248A,Y44V (Fig. 8, lanes c
and d) or R248A,E241S (Fig. 8, lanes e and
f) double mutants, the soluble pool of Ypt7p (Fig. 8,
center panel) completely shifted to the
membrane-bound form at both the permissive temperature and at the
restrictive temperatures (Fig. 2). A similar result was observed for
Vps21 (Fig. 8, bottom panel, compare a and
b to c and d). The distribution of
Ypt1p (Fig. 8, top panel) was more modestly affected at the
permissive temperature reflecting its apparent slower rate of
recycling. However, shift to the membrane-bound form was enhanced by
incubation at 37 °C reflecting inactivation of Sec19-1p (Fig. 8,
top panel, compare b to f). These
results are quantitated in Fig. 9. In particular, it is clear that
expression of the double mutants at levels similar to endogenous Gdi1p
pool interferes with the recycling of all Rabs from the membrane to the
cytosol. Thus, the double mutants, that are unable to bind Rab can
trigger a shift in the steady-state distribution of Rab from the
cytosol to the membrane even under permissive and semipermissive conditions where Sec19-1p is functional in growth. These results suggest that a Rab retrieval step is at least one target for the dominant effect of double mutants on growth. Moreover, given the fact
that binding of Gdi1p to membranes is independent or Rab, but dependent
on a trypsin-sensitive membrane-associated factor, this result provides
evidence for a novel membrane-associated receptor in these events.
GDI is critical for vesicular membrane transport in both the
endocytic and exocytic pathways. Our genetic analyses provide new
mechanistic insight into the GDI-Rab cycle. Using site-directed mutagenesis to inactivate Gdi1p function we have now established the
physiological importance of residues previously implicated in Rab3A
binding in vitro. These reveal a requirement for a novel recycling factor in the retrieval of Rab-GDP from membranes. The importance of our findings on the function of the Rab GTPase cycle (summarized in Fig. 10) is discussed in
detail below.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
isoform of GDI, first discovered by Takai and
colleagues (4) based on its ability to inhibit the intrinsic
dissociation of GDP from Rab3A, is now recognized to be one of 2 major
isoforms (
and
) found in mammalian cells that have nearly 85%
identity and are highly related to yeast Gdi1p (>50% identity with
-GDI), the product of the single, essential GDI1/SEC19
gene (5). Southern blot analyses of genomic DNA indicate that both
mouse and rat contain at least five rab GDI genes (6). GDI
family members are closely related to members of the CHM/REP gene
family involved in Rab prenylation (reviewed in Ref. 7), thereby
forming a GDI superfamily (reviewed in Ref. 3). While other Rab
effector proteins, including guanine nucleotide exchange factors or
GTPase activating proteins, exhibit specificity for individual Rab
proteins, GDI family members recognize all Rab proteins examined to
date (8, 9). This has led to the proposal that GDI/REP family members
principally function in maintaining a cytosolic reservoir of Rab
proteins for delivery to membranes.
-GDI at 1.81-Å resolution
using x-ray crystallography (10).
-GDI is constructed of two main
structural units, a large multisheet domain I and a smaller
-helical
domain II. Domain I is largely composed of sequence conserved regions
(SCRs) which are common to all members of the GDI superfamily. SCRs
located in the NH2-terminal and central portions of the
molecule fold to form a compact structural unit at the apex of GDI. In
particular, SCRs 1 and 3B contain tri- and tetrapeptide motifs that are
invariant from yeast to man (11). The polar side chains of amino acids
in these motifs are directed away from the
-carbon backbone,
suggestive of a role in the recognition of other proteins. Selected
residues in SCRs 1 and 3B have been implicated for the binding of Rab3A
in vitro and for the ability of
-GDI to extract the
GDP-bound forms of Rab3A from permeabilized rat brain synaptosomes
(10). Recently, we have found that residues involved in Rab recognition
have differential effects on Rab1 binding and ER to Golgi transport
in vitro (12). These residues, in addition to other highly
conserved residues distributed throughout the NH2-terminal
half and central half of GDI are grouped on only one face of the
molecule, leading us to previously speculate that this face is involved
in most, if not all, important features of GDI/CHM biological function
(13).
-GDI involved
in Rab3A binding and membrane extraction in vitro (10)
exhibit reduced Rab binding in vivo yet retained partial
Gdi1p function in support of growth and transport. These results
establish for the first time the physiological importance of the
putative Rab-binding platform found at the apex of GDI for function
in vivo and that a functional excess of GDI-Rab complexes
are present in living cells. Strikingly, selected residues involved in
Rab binding in vitro, when combined in double mutants, had
dominant effects on growth, were found to render GDI completely
defective in Rab binding in vivo, failed to complement
vesicular transport and growth defects of gdi1
and
gdi1 temperature-sensitive mutations, and led to the
accumulation of Rabs on membranes. Despite these changes, these double
mutants bound membranes as efficiently as wild-type Gdi1p in
vivo and in vitro. The implication of these results on the mechanism of GDI function in the delivery and retrieval of the
Rab-GDP from membranes following vesicle fusion reveals a potential
role for a rab recycling factor that mediates GDI-dependent retrieval of Rab-GDP.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain (CBY71) was constructed by transformation
of a wild-type diploid strain (SEY 6210a/
) with a CEN
URA3 plasmid containing GDI1, and then
transformation of this strain with the gdi1
::HIS3 deletion-disruption
construct (described below). After confirming by polymerase chain
reaction that this strain contained one intact and one disrupted copy
of GDI1, it was sporulated and colonies derived from
Ura+ His+ spores were identified. The
sec19-1 strain, CBY47 (sec19-1 ura3-52 trp1-
901
leu2-3, 112 his3-
200), was constructed by crossing NY420
(sec19-1 ura3-52) to a wild-type strain (SEY 6210),
sporulating this strain and identifying colonies which did not grow at
37 °C.
::HIS3 construct was generated by replacing
the DNA fragment encoding amino acids 17 through 414 of Gdi1p with a
DNA fragment containing the HIS3 gene. The gene SOEing
technique (22) was used to make gdi1 point mutants with DNA
oligonucleotides from Life Technologies, Inc. All mutants were
sequenced to confirm that only the intended mutations were present.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
null mutation. Plasmids (CEN
LEU2) containing site-directed mutations were used to
transform a gdi1
::HIS3 strain containing a CEN
URA3 GDI1 plasmid as the sole source of wild-type Gdi1p.
Transformed strains were streaked to 5-fluoroorotic acid plates to
select for loss of the URA3 GDI1 plasmid leaving the mutant
gdi1 gene as the only source of Gdi1p. Growth of the
resultant strains was assayed after restreaking to rich medium. This
method allowed us to assess if any gdi1 mutants constructed
were able to supply the essential function of GDI1. As a
test to confirm the feasibility of this approach, two separate point
mutations (I13R or T256P) were introduced into GDI1. Based on the crystal structure (10), we predicted that each of these point
mutations (Fig. 1) would affect the
structural integrity of the Rab-binding region of Gdi1p and lead to a
complete loss of function. As expected, each of these mutants failed to
yield Leu+ Ura
strains after streaking to
5-fluoroorotic acid plates, indicating non-complementation of the
gdi1
null mutation and thus confirming the validity of
this assay (Table I, single
asterisk).
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Fig. 1.
Location of substitutions (yeast numbering)
based on the crystal structure of bovine GDI
(10). The location of each of the residues mutated are indicated.
The insert region (light gray) and the Rab-binding region
(dark gray) containing SCRs 1B and 3B are highlighted at the
top of GDI. The location of helix I and N and
NH2 and COOH termini are indicated. The COOH-terminal 18 residues of GDI are disordered in the crystal structure of GDI and
therefore not shown (10). The conserved face of GDI containing SCRs 2 and 3A is oriented to the front of the image (10).
Effect of single point mutations in GDI on growth of the gdil strain
mutation (Table II). One lethal construct was a
triple mutant involving Arg248 and the residues
GXG potentially involved in the binding of a cofactor to GDI
(Table II, single asterisk) (10). Significantly, two of the lethal
double mutant combinations (Y44V,R248A and R248A,E241S) (Table II,
double asterisks) contained residues (Tyr44 and
Glu241) which when mutated separately, have been previously
implicated in Rab-binding in vitro (10). Thus, disruption of
the physiological function of Gdi1p in vivo requires
mutation of at least two surface residues in the putative Rab-binding
region (10). All other double mutant combinations complemented the
gdi1
mutation (Table II). Our results suggests that Rab
interacts with GDI through multiple residues and demonstrates for the
first time the physiological importance of the Rab-binding platform for
the function of GDI in living cells.
Effect of double mutants on growth of the gdi1 strain
mutation also did not complement the sec19-1 mutation at
37 °C, in complete accord with the results of the gdi1
complementation test (Fig. 2A). Notably, when the R248A,Y44V
and R248A,E241S double mutants were transformed into the
sec19-1 strain, we observed significantly slower growth at
26 °C and a dramatic dominant growth defect at 30 °C (Fig.
2B), a semipermissive temperature for sec19-1. Interestingly, neither of the double mutants caused a growth defect when introduced into a wild-type strain, indicating that the double mutants exhibit an allele-specific genetic interaction with
sec19-1. These results demonstrate that Gdi1p encoded by
sec19-1 is likely to be weakly defective for function at
both the permissive and semipermissive temperatures, and that this
phenotype is clearly exacerbated by competition with a double mutant
which fails to bind Rab (see below).
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Fig. 2.
Growth of the sec19-1 strain
expressing gdi1 point mutants. A, CBY47
(sec19-1) was transformed with a CEN vector containing the
indicated gdi1 point mutants. Transformants were maintained
at 26 °C, then streaked to selective plate and incubated at 37 °C
for 3 days. B, CBY47 (sec19-1) was transformed
with a CEN vector containing the indicated double gdi1 point
mutants and wild-type GDI1. Transformants were maintained at
26 °C, then streaked to selective media and incubated at 30 °C
for 3 days.
mutation or
the sec19-1 ts phenotype could produce proteins that are defective for folding and therefore unstable and degraded.
Alternatively, they could be deficient in any essential aspect of Gdi1p
function, including Rab binding or interactions with membrane receptors possibly required for Rab delivery or extraction. To distinguish between these possibilities, a vector was constructed which allowed us
to tag mutant and wild-type Gdi1p proteins with the influenza hemagglutinin (HA) epitope at the carboxyl terminus (HA-Gdi1p). This
region in bovine GDI is disordered in the crystal structure (10) and
the epitope tag would not be expected to interfere with Gdi1p function.
Consistent with this conclusion, all HA-tagged constructs generated
behaved in an identical fashion to their equivalent untagged
versions.2
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Fig. 3.
Co-immunoprecipitation of Rabs with wild-type
and mutant Gdi1 proteins. A and B, identical
amounts of cell homogenates of sec19-1 cells expressing HA
epitope-tagged wild-type (WT) or mutant Gdi1 proteins
(indicated at the top of each lane) were cleared of
membranes by centrifugation at 100,000 × g, and Gdi1p
was immunoprecipitated from each supernatant fraction (S100) under
native conditions. In A, HA-Gdi1p was detected using
immunoblotting with a HA-specific antibody in cell homogenates (before
immunoprecipitation), to show that identical amounts of HA-Gdi1p is
present in each sample. In B, antibodies to Ypt1p, Ypt7p,
and Vps21p were used to detect the corresponding proteins in the
immunoprecipitates using immunoblotting. C, identical
amounts of the pellet fraction of the 100,000 × g
centrifugation (P100) were either not washed (e-h) or
resuspended and washed (a-d) in an identical way to that of
the immunoprecipitations described for panel B. In
a and e, the P100 was prepared from the Sec19-1
strain containing the vector only; in b and f,
the P100 was prepared from cells expressing HA-wild-type Gdi1p; in
c and g, the P100 was prepared from cells
expressing HA-R248A,Y44A Gdi1p; in d and h, the
P100 was prepared from cells expressing HA-R248A,E241S. HA-Gdi1p was
detected using immunoblotting with an HA-specific antibody.
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Fig. 4.
Distribution of wild-type and mutant Gdi1
proteins between cytosol and membranes. A, strains
(sec19-1) expressing the indicated HA epitope-tagged Gdi1
proteins were lysed and subjected to centrifugation at 100,000 × g to generate a P100 membrane fraction and an S100 cytosol
fraction. Gdi1p in each fraction was visualized after immunoblotting
with the HA antibody. The vector only control is shown in lane g.
B, in vitro binding of HA-tagged wild-type and
R248A,E241S Gdi1 proteins to membranes. S100 fractions of the
sec19-1 strain expressing vector only (control)
(a and d), HA-tagged wild-type (b and
e), or R248A,E241S (c and f) were
isolated and incubated at 30 °C for 30 min with cell homogenates
(a-c) or P100 membrane fractions (d-f) prepared
from sec19-1 transformed with vector only. Subsequently, a
P100 fraction of each incubation condition was isolated, washed, and
probed with a HA-specific antibody using immunoblotting. C,
in vitro binding of HA-GDI to membranes is
trypsin-sensitive. A P100 membrane fraction prepared from
sec19-1 transformed with vector only was not treated
(a and d), treated with trypsin premixed with a
2-fold molar excess of trypsin inhibitor (b and
e) (1% weight/weight for 15 min on ice), or trypsin alone
(c and f). In c and f,
after trypsin digestion, a 2-fold molar excess amount of trypsin
inhibitor was added. Subsequently, P100 samples were mixed with
HA-Gdi1p (a-c) or the HA-tagged-R248A-E241S Gdi1p
mutant as described in B and incubated at 30 °C for 30 min. A P100 fraction of each sample was isolated at the end of the
incubation and probed with a HA-specific antibody using
immunoblotting.
strain
mentioned above. The sec19-1 strains with each of the three
single gdi1 mutant plasmids had more p2CPY present at the
10-min chase point compared with cells with wild-type GDI1.
At the 30-min chase point in Y44V, E241S, and R248A containing cells,
mCPY was the major form present. Notably, in R248A containing cells, a
small amount of p1CPY persisted even at the 30-min chase point
reflecting the reduced ability of this mutant to recognize Ypt1p (Fig.
3B). Overall, these results are fully consistent with the
growth phenotypes associated with each of the single gdi1
mutants (as the only source of Gdi1p).
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Fig. 5.
Analysis of vesicle-mediated trafficking in
gdi1 mutants. The indicated strains were prepared
for labeling at 26 °C, then the cultures were split and one-half was
transferred to 37 °C 10 min prior to labeling. Cultures were labeled
with [35S]methionine/cysteine for 10 min, then chased for
10 or 30 min. CPY was immunoprecipitated from extracts derived from
each culture. The positions of p1CPY (ER and Golgi), p2CPY (Golgi and
endosome), and mature CPY (vacuole) are indicated to the
left. B, spheroplasts of the indicated strains
were pulse-labeled for 5 min at 30 °C as in A, and then
chased (30 °C) for 0 or 5 min. Cells were collected by
centrifugation and Hsp150 was immunoprecipitated from the cell fraction
(I, intracellular) and from the media fraction
(E, extracellular).
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Fig. 6.
Endocytosis of the lipophilic dye FM4-64 in
gdi1 mutants. The indicated strains were prepared
at 26 °C, then shifted to 37 °C 10 min prior to addition of media
(37 °C) containing FM4-64. After 10 min, fresh media was added and
aliquots were removed immediately and at 45 min. The distribution of
FM4-64 was visualized by fluorescence microscopy.
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Fig. 7.
Electron microscopy of gdi1
mutant strains. sec19-1 (A and
C) and sec19-1 R248A,Y44V strains (B
and D) were grown overnight at 26 °C, then each culture
was divided and one-half was incubated at 37 °C for 1 h
(C and D), and the other half was maintained at
26 °C (A and B). An enlargement of a
sec19-1 R248A,Y44V at 37 °C field, highlighting the
numerous large vesicles which accumulate in this mutant, is shown in
E. For panels A-D, the scale bar
represents 1 micron, and in E the scale bar
represents 0.2 micron.
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Fig. 8.
Distribution of Rab proteins in
gdi1 mutant cells. The indicated strains
(sec19-1; sec19-1 with CEN plasmids encoding
gdi1 R248A,Y44V or R248,E241S) were labeled with
[35S]methionine/cysteine for 15 min, then chased (30 min)
at 26 °C. Each culture was divided in half and one aliquot was
transferred to 37 °C and the other returned to 26 °C, and the
cultures were incubated for 30 min. Cells were lysed and the resulting
extracts were centrifuged at 100,000 × g for 45 min to
generate membrane and cytosol fractions. Equal aliquots (5 OD units of
yeast) were treated with antibodies specific to the indicated Rab
proteins to quantitatively immunoprecipitate each of the Rabs that were
visualized by autoradiography. Because the majority of each Rab is
membrane-associated, autoradiograms of the cytosol fractions were
exposed 3 times as long as the autoradiograms of the membrane
fractions. All samples were processed and exposed to x-ray film in an
identical fashion.
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Fig. 9.
Quantification of the redistribution of Rab
from cytosol to membranes in the presence of double mutants. The
panels in Fig. 8 were quantitated using densitometry (Molecular
Dynamics). The values are reported as % of total Rab protein found in
the cytosol of the sec19-1 strain grown at the permissive
temperature (lane a).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
The potential role of a Rab recycling factor
(RRF) in Rab retrieval. In the cytosol, Rabs are
associated with GDI (bottom, middle). Membrane loading may
require a GDI displacement factor (GDF) (16). On transport
vesicles, the GTP-loaded Rab is required for vesicle docking and
fusion. After fusion, Rab-GDP is recycled to the cytosol through the
recruitment of GDI by a putative RRF.
The Rab-binding Region Plays a Physiological Role in GDI Function
in Vivo--
Previous biochemical studies based on the structure of
bovine -GDI led us to suggest that conserved residues present in
SCRs 1 and 3B fold to form a platform at the apex of GDI which
participates in the binding of mammalian Rab3A in vitro
(10). Mutations in this region of bovine
-GDI led to marked
reduction in the recognition of Rab 3A, inactivated the ability of GDI
to extract Rab proteins in vitro, and interfered with the
ability of GDI to inhibit ER to Golgi in vitro (10, 12).
Given the indirect nature of these previous in vitro
studies, we have characterized for the first time the physiological
importance of these and other residues in the function of yeast Gdi1p
in vivo. Single point mutations in yeast GDI1
resulted in mutant Gdi1p proteins that still complemented the
gdi1
mutation in vivo, although interactions
with Rabs were clearly affected (Fig. 3). The surprising ability of
mutant Gdi1 proteins with reduced affinity for Rab to support the
essential function of GDI1, lead us to suggest that in the
yeast Gdi1p-Rab recycling pathway, the cytosolic Rab pools bound to
Gdi1p are likely to be in functional excess. Consistent with this view, several Rab proteins were nearly exclusively membrane-associated in
sec19-1 cells expressing Gdi1p double mutant at the
permissive temperature (Fig. 8), yet growth and membrane trafficking of
these strains were largely unimpaired. Thus, only a limited recycling cytosolic pool is essential for Gdi1p function in cell growth. These
results suggest that in vitro biochemical assays of Rab binding by GDIs may not accurately reflect the ability of Gdi1p to
function in vivo.
In contrast to the lack of striking effects of single gdi1
point mutations on Gdi1p function, combinations of mutations in regions
of -GDI previously implicated in Rab binding in vitro (10) rendered yeast Gdi1p completely inactive in vivo
despite normal levels of expression and partitioning the protein
between the cytosolic and membrane-associated pools. In particular, the identification of double mutants defective in Gdi1p function stressed the importance of Arg248 (Arg240 in mammalian
-GDI). The combination of the R248A mutation with mutations in other
conserved residues of SCRs 1 and 3B, such as Y44V (Y39V in
-GDI) or
E241S (E233S in
-GDI), previously implicated in Rab3A binding
in vitro (10) were unable to support growth of the
gdi1
strain.
Arg248 is found in the center of helix I which forms the
front edge of the putative Rab-binding platform (10) (Fig. 1). Mutation of the equivalent residue in bovine -GDI (Arg240) led to
an ~60-fold reduction in the binding of Rab3A to
-GDI in
vitro and neutralized the ability of
-GDI to extract Rab3A from
synaptosome membranes in vitro. In the context of the
structure of
-GDI (10), our results now provide strong physiological evidence that Arg248 plays a pivotal role along with other
residues found at the apex of GDI to the formation of the functional
Rab-binding "platform" (12).
Distribution of Rabs Is Differentially Sensitive to Residues in the
Rab-binding Platform--
Our studies have demonstrated that the
binding and distribution of different Rab proteins between membrane and
cytosolic fractions were differentially sensitive to mutations in
GDI1. One explanation for this result is that residues in
the Rab-binding pocket of Gdi1p contribute in different ways to the
strength of interaction between Gdip and distinct Rab species, a result
consistent with in vitro binding studies involving -GDI
and mammalian Rabs (12). A second possibility is that the differential
effects of Gdi1p inactivation on Rab distribution could reflect, in
addition to differences in Rab recognition, the overall kinetics of
Gdi1p-dependent recycling. Recycling per se
could be dependent on multiple factors including the amount of membrane
being converted to vesicular carriers per unit time and the
steady-state abundance of each Rab GTPase in the cell. It has been
shown that a permanently membrane-associated mutant Ypt1 protein (with
COOH-terminal isoprenylation sites substituted with a transmembrane
domain) can fulfill the essential function of Ypt1p in ER to Golgi
transport, albeit inefficiently (36). This led to the suggestion that a
cytosol-dependent translocation step is not absolutely
required for Ypt1p function. However, our results now demonstrate that
only a very small pool of Rab is required for function. Therefore, even
an ER-associated reservoir of newly synthesized Ypt1 may have
contributed significantly to the success of this experimental approach
(36). We suggest that GDI-dependent translocation through
the cytosol is essential for Rab function.
Morphological Effects of GDI Mutants--
After a brief incubation
of sec19-1 at the restrictive temperature, we noted an
accumulation of aberrant membranes, but few transport vesicles (Fig.
7). These results were surprising in light of the results of Garret
et al. (5) who found that gradual depletion (over 15 h)
of wild-type Gdi1p led to accumulation of ER, Golgi-like elements, and
transport vesicles. The distinction between these experiments and ours
(gradual depletion of functional Gdi1p versus rapid
inactivation of Gdi1p) is important because it revealed a novel
requirement for Gdi1p in the function of exocytic and endocytic
compartments. One possibility is that Gdi1p is required for vesicle
biogenesis. This conclusion is supported by recent experiments that
have shown a requirement for GDI-Rab5 in clathrin-mediated endocytic
vesicle formation (37). In addition, in vitro experiments following the requirement for Rab1
(38)4 and analysis of
ypt31 ypt32
yeast mutants (39) indicates that Rab proteins are required for vesicle budding from the ER and
Golgi, respectively. Alternatively, Rab-dependent homotypic fusion is now recognized to play a prominent role in the architecture of the ER, Golgi, and endosome. It is possible that the novel structures detected morphologically following rapid turn-off of Gdi1p
function represent defects in these events.
Rab Delivery to Membranes Requires GDI and Binding of GDI to
Membranes Is Rab-independent--
While a variety of in
vitro experiments have implicated an important role for the
delivery of Rab to membranes through a cytosolic GDI·Rab complex
(reviewed in Refs. 2 and 3), this point remains to be established
physiologically. Our ability to detect a prominent cytosolic
Sec19-1p-Rab pool under conditions in which Sec19-1p was rapidly
rendered dysfunctional (Fig. 8) is consistent with this assumption.
Importantly, we demonstrated that the association of Gdi1p with
membranes was independent of its ability to bind Rab (Figs. 3 and 4).
Thus, while targeting of the GDI-Rab to specific compartments is
believed to involve the hypervariable carboxyl terminus of different
Rab species (40), we now suggest that stable membrane association of
Gdi1p is Rab-independent. Consistent with this possibility, we have
recently shown that mutation of Arg70 in bovine -GDI
resulted in loss of Rab1 binding, yet the
-GDI mutant bound
membranes strongly and was a potent inhibitor of transport of protein
between the ER and Golgi (12). One possible candidate receptor is
"GDI displacement factor" or guanine nucleotide displacement
factor, a protein suggested by Pfeffer and colleagues (14, 16) to
promote release of endosomal Rab GTPases from the Rab·GDI complex
during Rab delivery to membranes. Alternatively, receptors that
function in other steps in the Rab GTPase cycle may be responsible for
stable binding.
Rab Recycling Factors Are Required for GDI Function in Vivo-- An important and unexplored aspect of Gdi1p function regards the molecular mechanism by which Gdi1p extracts Rabs from membranes. GDP-bound Rabs to be extracted from membranes could potentially serve as Gdi1p "receptors" and recruit free Gdi1p from the cytosol. Our in vivo and in vitro results, however, indicate that Rab binding is not exclusively responsible for the association of Gdi1p with membranes. Moreover, we noted that the sec19-1 strain containing the double mutant, when incubated at the permissive or restrictive temperatures, led to the enhanced redistribution of multiple Rabs from the cytosol to membranes despite the fact that the double mutant could not recognize Rabs. The residual cytosolic pools, in the case of Ypt1p, may reflect weak binding to either misfolded Sec19-1 Gdi1p or the double mutant that cannot be detected in vitro. The effect of the double mutant on steps involved in Rab recycling in vivo was corroborated with its observed ability to efficiently bind membranes in vitro, even in the presence of Sec19-1p.
One possibility to explain our results is that Gdi1p or Gdi1p·Rab
complex functions as a multimer and the mutant Gdi1p interferes with
this function. We feel that this explanation is unlikely given that all
experimental evidence to date demonstrates that GDI is found in the
cytosol as a monomer or as a heterodimer with Rab (3). An alternative
explanation that we prefer is that our findings now imply the activity
of a novel membrane-associated factor(s) that mediates the recruitment
of Gdi1p for Rab-GDP recycling steps. This factor, referred to as
Rab recycling factor or RRF (Fig.
10), we would propose directs the interaction of unbound Gdi1p with
newly formed Rab-GDP species formed during or following membrane
fusion. Consistent with this interpretation, we have observed a
requirement for a membrane-associated factor to recruit mammalian
-GDI in ER to Golgi transport in vitro (12), although we
were unable to assess whether it operated in Rab delivery or Rab
retrieval. Our proposal for a requirement for RRF in the retrieval of
Rab-GDP is supported by the observation that double mutant Gdi1
proteins interfered with recycling of Rabs by Sec19-1p at both the
permissive and restrictive temperatures, directing a shift in the
steady-state distribution from the cytosol to membranes (Fig. 8).
Moreover, the double mutant Gdi1p also caused dominant growth defects
in the sec19-1 background at the semipermissive temperature
(Fig. 2), suggesting that a recycling factor(s) that is not a Rab
becomes inaccessible to the partially functional sec19-1
Gdi1p pool, possibly through competition by the double mutant.
Of related interest to the above observations is that overexpression of
wild-type Gdi1p in yeast or -GDI in mammalian cells does not have
the same potent inhibitory effect on ER to Golgi or intra-Golgi vesicle
transport in vivo as is observed when permeabilized cells
are preincubated with excess GDI in vitro (8,
41-43).5 However, other Rab
proteins, such as Rab11 that mediates transport of vesicular stomatitis
virus glycoprotein from the trans Golgi network to the cell surface, is
very sensitive to GDI overexpression (41). These results, combined with
a potential requirement for RRF, raises the possibility that
membrane-associated Rabs may be restricted to the GTP-bound form
in vivo when bound to membranes and inaccessible to GDI
removal except at a defined point in the Rab cycle when it encounters
RRF during or following vesicle fusion. The relationship between these
observations and the finding that Rab5 undergoes rapid GTP turnover
in vitro could reflect reduced stability of Rab5 (GTP) under
reconstitution conditions (44). The implications of our observations
for the model that Rab functions as a molecular timer in
vivo remains to be clarified (44). We are currently exploring the
identity of residues in Gdip involved in recognition of RRF by
extending our current mutagenesis to residues in other SCRs such as 2 and 3A that line the conserved face of GDI.
Implications for Physiological Function of the GDI
Family--
While a variety of studies have shown little difference in
the ability of various GDI isoforms to distinguish between different Rabs (8, 9, 43, 45, 46), it is known that the expression level of a
particular GDI isoform is highly variable between tissues. The
-isoform of GDI is nearly exclusively found in brain tissue, whereas
the
isoform is ubiquitously expressed (47, 48). The altered levels
of tissue distribution of GDI isoforms combined with our observations
that residues in the Rab-binding region may contribute to differential
activity in Rab recognition (and recycling), leads us to suggest that
GDI isoforms play specialized roles in the handling of subsets of
tissue-specific Rabs. Such specialization may contribute to higher
efficiency of specific types of endomembrane traffic in these tissues.
Support for this proposal comes from our recent discovery in
collaboration with Toniolo and colleagues (49) that the
-GDI isoform
of GDI is responsible for X-linked mental retardation. Patients who are null for
-GDI are phenotypically normal with the exception of reduced mental capacity, implying an important role for
-GDI in the
recycling of Rab3 GTPases in the development of the synapse responsible
for human intelligence.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Peter Novick and William Synder for generously providing reagents, Peggy Mustol for assistance with strain construction, and Tammy McQuistan and the Electron Microscopy Core Facility for electron microscopy services (core B of CA58689).
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM33301 (to W. E. B.) and Core B in CA 58689 (to S. D. E.).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. E-mail: webalch{at}scripps.edu.
Investigator of the Howard Hughes Medical Institute.
** Supported as a Postdoctoral Fellow of the American Cancer Society.
2 P. Luan, W. E. Balch, S. D. Emr, and C. G. Burd, unpublished observations.
3 P. Luan, W. E. Balch, S. D. Emr, and C. G. Burd, unpublished results.
4 W. E. Balch, unpublished data.
5 W. E. Balch, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: GDI, guanine nucleotide dissociation inhibitor; SCR, sequence conserved region; ER, endoplasmic reticulum; HA, hemagglutinin; CPY, carboxypeptidase Y.
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REFERENCES |
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