Identification of Ypt1p-GEF
To identify a GEF activity for Ypt1p in yeast cells, we first assayed the ability of cell lysates to stimulate release of GDP from Ypt1p. Crude extracts of Saccharomyces cerevisiae strain GPY60 and fractions obtained by differential centrifugation were incubated with recombinant Ypt1p preloaded with [3H]GDP. Yeast cell extracts stimulated GDP release from Ypt1p above the intrinsic rate in a time- (Figure A) and concentration-dependent manner. Because we later show that this activity also promotes GTP uptake specifically by Ypt1p (see below), we refer to this activity as Ypt1p-GEF. Comparing equal amounts of protein from different cell fractions revealed that the P100 fraction was enriched in GEF activity, whereas there was no detectable or little activity in the S100, S12, and the P12 fractions, respectively (Figure A). The highest specific activity of Ypt1p-GEF was found in the P100 fraction (~three- to fivefold enrichment relative to the crude lysate; Figure B). Approximately one-half of the Ypt1-GEF activity present in the total extract was recovered in the P100 fraction, whereas very little (~2.5%) was found in the P12 fraction (Figure C), suggesting that the Ypt1p-GEF associates with a light particulate cellular compartment.
Figure 1 Identification of a GDP release-stimulating activity for Ypt1p in the P100 fraction of yeast cells. (A) Time course of GDP release from Ypt1p in the presence of fractionated yeast extracts is shown. Ypt1p-[3H]GDP was incubated in the presence (more ...)
To characterize the association of the Ypt1p-GEF activity with this particulate compartment, we extracted the P100 fraction with detergents and NaCl. Although Ypt1p can be extracted from the P100 fraction by nonionic detergent treatment, the Ypt1p-GEF activity was resistant to detergent treatment. Instead, ~75% of the GEF activity was liberated by 0.5 M NaCl (Figure A). Increasing the NaCl concentration beyond 0.5 M did not significantly increase the recovery of Ypt1p-GEF activity. The ability of the Ypt1p-GEF to be extracted by salt but not by detergent is consistent with an association with membranes or cytoskeletal elements by electrostatic interactions.
Figure 2 Association of the Ypt1p-GEF with the P100 fraction and preparation of the solubilized GEF fraction. (A) Extraction of Ypt1p-GEF from the P100 fraction by salt but not by detergents is shown. P100 was treated with 1% Triton X-100, 30 mM n-octylglucoside, (more ...)
Salt extraction of Det-P100 yielded solubilized Ypt1p-GEF activity free of Ypt1p. The solubilized GEF activity is enriched 4-fold relative to the P100 fraction and ~16- to 20-fold relative to the total cell extract (Figure B). The solubilized GEF was proteinaceous because it was sensitive to protease or heat but not to RNase or DNase. The Det-P100 fraction was used for experiments in which high concentrations of the exchange activity were needed, whereas the salt extract was used for further purification of the Ypt1p-GEF (see below; the solubilized GEF fraction could not be used in high concentrations because salt interferes with the exchange reaction and salt removal caused protein precipitation).
To confirm that this Ypt1p-GEF activity corresponds to a genuine exchange activity, we used the partially purified exchange factor to assay stimulation of GTP binding by recombinant Ypt1p. Recombinant Ypt1p was preloaded with nonradioactive GDP; then the binding of [α-32P]GTP was measured in the presence of either the Det-P100 fraction or the solubilized GEF. The intrinsic rates of GDP release and GTP uptake of Ypt1p were very similar (0.76 ± 0.13 and 0.87 ± 0.12 fmol/min per picomole of Ypt1p, respectively). Adding increasing amounts of the Det-P100 fraction accelerated GTP uptake by Ypt1p to a maximum 10-fold stimulation at 5 mg/ml (Figure A). When the solubilized GEF was used as a source of exchange activity, both GTP uptake and GDP release rates were also linear with respect to concentration and time (Figure B) and were essentially identical. Thus, the activity that we identified on the yeast cell membranes and that was solubilized and partially purified stimulates the exchange of Ypt1p-bound GDP for GTP at least 10-fold.
Figure 3 Stimulation of GTP uptake by Ypt1p-GEF. (A) Increasing concentrations of the Det-P100 (Triton X-100–extracted) fraction result in increasing stimulation of GTP uptake by Ypt1p. Ten picomoles of Ypt1p preloaded with nonradioactive GDP were incubated (more ...)
A candidate for Ypt1p-GEF activity is Dss4p, a suggested GDP-release factor for the closely related Sec4p. Previous work demonstrated that purified recombinant Dss4p was capable of stimulating release of GDP from Ypt1p by 2.5-fold above the intrinsic rate (Moya et al., 1993
). We compared the stimulation of GDP release for Ypt1p by yeast cell extracts prepared from a wild-type strain and a strain in which the DSS4
gene was deleted (Moya et al., 1993
). P100 fractions prepared from both yeast strains yielded equivalent results and stimulated GDP release from Ypt1p by 4- to 5-fold (Table A). Therefore, the Ypt1p-GEF activity that we have identified is not Dss4p.
Activity of Ypt1p regulators in extracts of mutant strains
Substrate Specificity of Ypt1p-GEF
To determine the specificity of the Ypt1p-GEF activity, we assayed the ability of the Det-P100 fraction to stimulate the exchange of guanine nucleotides bound to Ras2p, Ypt32p, or Sec4p. Under conditions in which the Det-P100 fraction stimulated GDP release by ~12-fold from Ypt1p, there was an ~4- to 6-fold stimulation of GDP release from Ras2p but no effect on the other two proteins (Figure ). To determine whether the exchange factors for Ypt1p and Ras2p are distinct, we used a mutant Ypt1p, D124N, that inhibits the Ypt1p-GEF (Jones et al., 1995
). Stimulation of nucleotide exchange by the Ypt1p was abolished by the mutant protein, while the stimulation of GDP release from Ras2p was unaffected (Figure , compare panels A and D), indicating that the factors that stimulate release of GDP from Ypt1p and Ras2p are distinct. Thus, the exchange activity present in the Det-P100 cellular fraction seems to be specific for Ypt1p and does not act on the other exocytic Ypt proteins. Partially purified Ypt1p-GEF (see HAP peaks A and B below) also failed to stimulate nucleotide exchange for Ypt31p and Ypt32p (our unpublished observations).
Figure 4 The Ypt1p-GEF activity present in the Det-P100 (Triton X-100– or n-octylglucoside–extracted) fraction does not act on Ypt32p or Sec4p and is different from the exchange factor for Ras2p. Ten picomoles of Ypt1p-[3H]GDP (A), (more ...)
At least one Rab GEF was reported to act preferentially on the prenylated form of Rab relative to the unprenylated form (Miyazaki et al., 1994
). However, prenylation of the recombinant Ypt1p had no effect on the ability of the Ypt1p-GEF (P100 fraction) to stimulate nucleotide exchange (our unpublished observations).
Partial Purification of Ypt1p-GEF
To purify Ypt1p-GEF further, the P100 fraction was extracted with 1% n-octylglucoside (because it is more readily removed by dialysis than is Triton X-100), and the residual membranes were extracted with 0.5 M NaCl to generate a solubilized GEF fraction that lacks Ypt1p. Sephacryl S-300 HR gel filtration partially resolved two peaks of activity with apparent molecular sizes of ~400–450 and ~200 kDa (Figure A). The two peaks were collected as individual pools, termed pool A and pool B. Approximately 17–31% of the starting Ypt1p-GEF activity was recovered in pool A with a purification of ~3- to 5-fold. Approximately 14–17% of the starting Ypt1p-GEF activity was recovered in pool B with a purification of ~1.5- to 1.7-fold. The apparent molecular sizes of the Ypt1p-GEF peaks were the same regardless of whether n-octylglucoside or Triton X-100 was used in the extract preparation and did not change if the solubilized GEF fraction was dialyzed to remove residual n-octylglucoside before chromatography. Thus the apparent molecular sizes derived from gel filtration are not attributable to protein inclusion in detergent micelles. Each S-300 pool was loaded separately onto a ceramic HAP column and eluted with a 10–200 mM potassium phosphate gradient. Pool A exhibited a peak of activity at ~105 mM phosphate, whereas pool B showed a single peak of activity at ~80 mM phosphate (Figure B). We verified that these partially purified activities, which stimulate GDP release from Ypt1p, are GEFs by assaying both pools for stimulation of GTP uptake. The specific activities measured by GDP release and GTP uptake were similar (within a factor of 2) during each step of the purification procedure. The purification factor after HAP chromatography was ~120 for peak A and ~37 for peak B. It is not clear whether the two peaks represent two different Ypt1p-GEFs or the same GEF in two different protein complexes.
Figure 5 Partial purification of the Ypt1p-GEF. (A) Gel filtration column. Two partially overlapping Ypt1p-GEF peaks are collected into distinct pools. The solubilized GEF fraction (Det-P100 extracted by salt) was separated on a Sephacryl S-300 HR column. Stimulated (more ...)
Rescue of ER-to-Golgi Transport by Partially Purified Ypt1p-GEF
We showed previously that the Ypt1p-GEF activity present in the P100 or Det-P100 fraction is completely inhibited by the Ypt1p-D124N mutant protein (Figure A; Jones et al., 1995
). The two peaks of Ypt1p-GEF generated by sequential purification on the S-300 and HAP columns are also inhibited to equal extents by Ypt1p-D124N (our unpublished observations). We have also shown that this dominant-mutant Ypt1p is a potent inhibitor of an ER-to-Golgi in vitro transport assay, probably because of inhibition of the Ypt1p-GEF (Jones et al., 1995
). To lend support to the assertion that the partially purified Ypt1p-GEF described here is an authentic Ypt1p-GEF, we tested the most purified exchange factor, peak A from the HAP column, for its ability to restore transport function to an in vitro ER-to-Golgi transport reaction inhibited by Ypt1p-D124N. The HAP peak A restored ~50% of the inhibited transport reaction in a concentration-dependent manner (Figure ). These results suggest that the partially purified exchange activity described here is a physiological Ypt1p-GEF.
Figure 6 Inhibition by Ypt1p-D124N of the in vitro transport reaction is relieved by Ypt1p-GEF, HAP peak A. The indicated amounts of exchange activity from HAP Pool A (1 unit is defined as 1 fmol of GDP released from 0.1 μM Ypt1p per minute per milligram (more ...)
Identification and Characterization of a GAP Activity for Ypt1p
GAP activity was measured as the stimulation of GTP hydrolysis by recombinant Ypt1p preloaded with [32
P]GTP, using the charcoal-binding method. Wild-type Ypt1p hydrolyzed GTP at a low intrinsic rate of 0.002 mol of GTP per mole of Ypt1p per minute at 30°C (Richardson et al., 1998
), similar to previously reported values (Wagner et al., 1987
). A GAP activity that stimulated GTP hydrolysis by Ypt1p was found to be highly enriched in the P12 fraction (see below). Treatment of this fraction at 95°C for 5 min or incubation with trypsin (1 mg/ml) ablated GAP activity, suggesting that the active factor is proteinaceous. GAP activity in the P12 fraction was linear with respect to protein concentration (0.5–1 mg/ml; Figure A) and time (0–30 min). Stimulation of hydrolysis approached a maximum at 5 mg/ml P12, in which the rate of GTP hydrolysis was increased 54-fold (to 0.108 mol per mole of Ypt1p per minute). Phosphate release from GTP hydrolysis in the charcoal-binding assay has been confirmed by TLC analysis that shows that the GTP is being converted to GDP. Two observations argue against the possibility that the GAP activity in the P12 fraction is a protease. 1) Ypt1p mutants that are resistant to the GAP activity have been identified (see below), and 2) the total amount of nucleotide bound to Ypt1p during the course of incubation with the P12 fraction remains constant.
Figure 7 Identification of a Ypt1p-GAP activity in the P12 cellular fraction and its solubilization. (A) GAP activity is linear with P12 concentration. Ypt1p (2 nM) preloaded with [γ-32P]GTP was incubated with the indicated quantities of (more ...)
To test models of Ypt/Rab function that assign the site of action of their GAPs to the acceptor compartment, we wanted to determine the compartment in which Ypt1p-GAP resides. Lysates were fractionated by differential centrifugation into 12,000 × g supernatant (S12) and pellet (P12) fractions. The S12 was then subjected to centrifugation at 100,000 × g to generate supernatant (S100) and pellet (P100) fractions. Although detectable Ypt1p-GAP activity exists in crude lysates, 73% of the activity fractionated into the P12 fraction where it is approximately eightfold enriched over that in the cell lysate (Figure B). Eighteen percent of the activity was found in the P100 fraction, where its specific activity is lower than is that in cell lysates. Therefore, the activity in the P100 probably represents contamination by P12 membranes. Only 1% or less of Ypt1p-GAP activity was found in the S100 fraction. The finding that the Ypt1p-GAP is enriched in the P12 fraction, with the majority of its activity found in this fraction, suggests that it is associated with large particulate cellular structures.
Extraction of Ypt1p-GAP from Membranes
The association of Ypt1p-GAP with P12 membranes was examined using different procedures, including incubation with trypsin, high salt, high pH, and detergent. Only limited trypsin digestion (0.1 mg/ml, 1 h, 0°C) resulted in solubilization accompanied by a two- to threefold stimulation of GAP activity (Figure C). This indicates that Ypt1p-GAP is tightly associated with P12 membranes. The trypsin extraction may lead to elevated GAP activity by increasing substrate accessibility.
One interesting feature of the Ypt1p-GAP is that it is potently inhibited by both ionic (CHAPS) and nonionic (n
-octylglucoside, Triton X-100) detergents. Triton X-100 causes a dose-dependent inhibition of GAP activity, and the trypsin-solubilized GAP is ~10-fold more sensitive than is the insoluble GAP (Figure D). Ypt1p itself is still active for GTP binding under these conditions (Richardson et al., 1998
). The lower sensitivity of the insoluble activity may be attributable to titration of Triton by membrane lipids and proteins. The detergent sensitivity of GAP does not appear to be attributable to interaction of GAP with the prenyl group of Ypt1p, because geranylgeranylated Ypt1p is as sensitive to detergent as unprenylated Ypt1p. Therefore, our data favor a model in which detergents exert their inhibitory effects either by unfolding GAP or by preventing interaction of GAP with Ypt1p.
Substrate Specificity of Ypt1p-GAP
The substrate specificity of GAP was examined using competition assays. To determine whether GAP has a higher affinity for the GTP- or GDP-bound form of Ypt1p, we incubated the P12 fraction with Ypt1p preloaded with [32P]GTP, and increasing concentrations of Ypt1p preloaded with cold nucleotide were added as a competitor. Ypt1p preloaded with either GppNHp (a poorly hydrolyzable analog of GTP; Figure A) or GTP were both effective competitors, but high concentrations (60 μM) were needed for 50% inhibition of GAP activity. This may reflect a relatively low-affinity interaction between GAP and recombinant Ypt1p. When Ypt1p preloaded with GDP is used as a competitor, ~60 μM gives only 32% inhibition of GAP activity. These data indicate that the GAP has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form.
Figure 8 Substrate specificity of Ypt1p-GAP. (A) Ypt1 GAP has a higher affinity for the GTP form than for the GDP form of Ypt1p. Ypt1p (2 nM) preloaded with [γ-32P]GTP was mixed with the indicated concentrations of competitor Ypt1p preloaded (more ...)
Because the P12 fraction may contain other GAP activities, we used competition assays to determine whether other Ypt proteins can compete with Ypt1p for the GAP activity. In their GppNHp forms, Ypt31p or Ypt32p, which share 42% identity with Ypt1p, were poor competitors for the GAP activity. On the other hand, Sec4p, which shares 48% identity with Ypt1p, showed 35% inhibition of GAP activity at 60 μM, as compared with 50% inhibition by Ypt1p itself (Figure B). Therefore, the Ypt1p-GAP is specific to a subgroup of exocytic Ypt proteins.
To assess the effect of mutations predicted to affect interactions of GTPases with their GAPs, we examined whether mutants of Ypt1p would exhibit reduced responsiveness to the activity. Specifically, we examined the behavior of a Ypt1p variant containing a mutation, T40K, in the putative effector domain, a region that is important for interaction of Ras with Ras-GAP (Boguski and McCormick, 1993
; Polakis and McCormick, 1993
). To determine whether Ypt1p-T40K is resistant to GAP, we assayed stimulation of GTP hydrolysis by wild-type or mutant Ypt1p preloaded with [32
P]GTP in the presence of the P12 fraction. As seen in Figure C, the intrinsic rate of GTP hydrolysis by Ypt1p-T40K is nearly identical to that of wild type. However, the mutant protein has an ~60% lower rate of GAP-stimulated hydrolysis. Therefore, the effector-domain mutation T40K impairs the ability of Ypt1p to respond to its GAP. GTP hydrolysis in the presence of GAP was found to be even more severely impaired in another mutant, Ypt1p-Q67L (Richardson et al., 1998
). These data support the suggestion that the activity we have identified does not cause the nonspecific dissociation and breakdown of GTP from Ypt1p but is an authentic Ypt1p-GAP.
Is Ypt1p-GAP the Product of a Previously Characterized Gene?
Several yeast proteins are known to act as GAPs for Ypt proteins (Strom et al., 1993
; Vollmer and Gallwitz, 1995
). Although Gyp6p was reported not to have GAP activity on Ypt1p (Strom et al., 1993
), possible activity of Gyp7p on Ypt1p was not reported. In addition, we found a closely related gene in the S. cerevisiae
genome database (YOR070C
), whose product was recently shown to have a GAP activity for Sec4p and Ypt1p and was termed GYP1
(Du et al., 1998
). To test whether Gyp7p or Gyp1p are responsible for the Ypt1p-GAP activity that we have identified, we deleted GYP7
individually or together. No reduction in GAP activity was observed in the P12 (or S12) fraction from either the single (our unpublished observations) or double deletion strains (Table B). Hence, the GAP activity we have characterized is the product of a gene or genes distinct from the known GYP
Among the proteins implicated in the ER-to-Golgi transport step in yeast are components and regulators of the soluble N
-ethyl-maleimide sensitive factor attachment protein (SNAP) receptor (SNARE) complex, a set of proteins thought to help determine the specificity of vesicle targeting. If Ypt1p regulates the assembly of the SNARE complex in a GTP-dependent manner, as suggested in the literature (Lian et al., 1994
; Sogaard et al., 1994
; Lupashin et al., 1996
), the assembled SNARE complex could turn off Ypt1p function by acting as a GAP or by signaling to a GAP. To test this, we determined whether preventing or promoting SNARE complex assembly would affect Ypt1p-GAP activity. To prevent SNARE complex assembly, we used extracts from sec22
mutant cells. SEC22
encodes a component of the SNARE complex, and sec22
mutants fail to form stable SNARE complexes (Lian et al., 1994
; Sogaard et al., 1994
). Conversely, to promote SNARE complex assembly, we generated extracts from sec17–1
cells, in which the SNARE complex is stabilized (Sollner et al., 1993
; Sogaard et al., 1994
). The cells were shifted to the nonpermissive temperature of 37°C for 1 h before cell lysis, and all cellular fractions were tested for GAP activity, in case inactivating one of these gene products caused redistribution of the GAP activity. No differences in the localization or specific activity of GAP were observed in fractions prepared from sec18–1
, or sec22–3
cells (Table C); thus, it is unlikely that the Ypt1p-GAP is either the product of these genes or influenced by the activity of these gene products.