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© The Rockefeller University Press,
0021-9525/2000//289 $5.00
The Journal of Cell Biology, Volume 151, Number 2,
, 2000 289-296
Original Article |
Trapp Stimulates Guanine Nucleotide Exchange on Ypt1p
TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.
Key Words: exchange factor • small GTPase • secretion • ER-to-Golgi • tethering factor
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
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Ypt/Rab proteins are molecular switches that cycle between GTP- and GDP-bound forms (Novick and Zerial 1997). This cycle is achieved by nucleotide exchange and GTP hydrolysis. Since the intrinsic rates of nucleotide exchange and GTP hydrolysis are low for most small GTP-binding proteins, accessory proteins are needed to stimulate these reactions. Guanine nucleotide exchange factors (GEFs) promote GDP dissociation and GTP uptake (reviewed in Martinez and Goud 1998), while GTPase-activating proteins stimulate GTP hydrolysis (reviewed in Scheffzek et al. 1998). In the case of Ypt1p, nucleotide exchange stimulated by a GEF is essential for its function (Jones et al. 1995). Ypt1p locked in its nucleotide-free state is a dominant negative inhibitior of ER-to-Golgi transport in vitro. Although previous studies have shown that Golgi membranes have a nucleotide exchange activity that acts on Ypt1p, this GEF has not been identified (Jones et al. 1998). In this report, we demonstrate that TRAPP binds to the nucleotide-free form of Ypt1p to accelerate nucleotide exchange on Ypt1p.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of Mutant Lysates
Cell lysates were prepared from the following strains: SFNY 26-3A (MATa ura3-52), SFNY596 (MATa bet3-1 leu2-3, 112 ura3-52), SFNY713 (MAT
GAL+ ura3-52 leu2-3,112: (LEU2 bet5-1) his3
200 bet5::HIS3), SFNY293 (MAT uso1-1 ura3 trp1 his7), SFNY691 (MAT sec34-2 ura3-52 leu2-3, 112), SFNY816 (MATa sec35-1 ura3-52), SFNY974 (MATa ura3-52 leu2-3, 112 his3200 trs13033::HIS3) referred to as trs130ts2 in the text, SFNY1019 (MAT ura2-52 trs31-1), and SFNY1040 (MATa ura3-52 trs85::URA3). Wild-type and mutant cells were grown overnight at 25°C to OD599 = 1.0. The cells (600 OD599 units) were converted to spheroplasts (Kim, et al. 1999) and lysed in 6 ml of buffer A (20 mM Hepes, pH 7.2, 1 mM DTT, protease inhibitor cocktail). Unbroken cells were removed during a 3-min centrifugation at 450 g and the supernatant (total lysates) was assayed.
In Vitro Binding Assay
Glutathione-S-transferase (GST)-Ypt1p and GST-Ypt51p were purified from 1.5L of Escherichia coli (DH5). Expression was induced for 2.5 h by the addition of IPTG (1 mM). The fusion protein was bound to a 2-ml bed volume of glutathione-agarose (Amersham Pharmacia Biotech) according to the manufacturer's protocol and stored in buffer I (50 mM Hepes, pH 7.2, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 mM MgCl2). Nucleotide was removed or exchanged on 200 µg of GST-Ypt1p or GST-Ypt51p (
4.1 nmol of Ypt protein assuming molecular mass of 48,000 for the fusion protein) in 500 µl of buffer I with a threefold molar excess of either GDP or GTP
S. To prepare the nucleotide-free forms of GST-Ypt1p and GST-Ypt51p, 10 mM EDTA was used in place of MgCl2 in buffer I. The beads were rotated at room temperature for 1 h and then washed two times with 1 ml of buffer II (50 mM Hepes, pH 7.2, 100 mM NaCl, 1 mM DTT, 5 mM MgCl2). To rebind GDP to the nucleotide-free form of GST-Ypt1p, the beads were washed two times with 1 ml of buffer I and incubated for 1 h with a threefold molar excess of GDP in 500 µl of buffer I. The beads were then washed two times with 1 ml of buffer II and immediately incubated with a yeast lysate as described below.
A lysate from wild-type yeast (SFNY26-3A) was prepared by converting 7500 OD599 units of cells to spheroplasts during a 1-h incubation at 37°C (Sacher et al. 2000) and lysing the cells in 140 ml of 20 mM Hepes, pH 7.2, with a Wheaton dounce homogenizer. The lysate was centrifuged at 25,000 rpm in a 70Ti rotor (Beckman) for 1 h and protein was measured by the Bradford assay. The lysate was then dialyzed overnight against buffer II. A total of 500 mg of lysate was incubated with GST-Ypt1p or GST-Ypt51p, prepared as described above, for 2 h at 4°C. The beads were then transferred to an Eppendorf tube following a 3-min centrifugation at 1500 g and washed two times with 1 ml of buffer II (±GDP or GTPS as required) and once with 1 ml of buffer II containing 250 mM NaCl. The beads were boiled in 150 µl of SDS-PAGE sample buffer and fractionated on an SDS-12.5% polyacrylamide gel. Western blots were probed with antibodies against the TRAPP subunits as described in the legend to Fig. 1.
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ura3-52 bet3::URA3 leu2-3, 112 BET3-protein A::LEU2 L-A-o) was used for the purification because it contained protein A–tagged Bet3p and was free of the L-A virus (L-A-o). The L-A virus coat protein, gag, is a common contaminant in the purification (see Sacher et al. 2000). To purify TRAPP, cells were converted to spheroplasts, lysed in 3 ml of buffer B (150 mM KCl, 20 mM Hepes, pH 7.2, 2 mM EDTA, 1% Trition X-100, 0.5 mM DTT, protease inhibitor cocktail), and the unbroken cells were removed as described above. The lysate was centrifuged at 14,000 g for 10 min and the supernatant (10 mg/ml of protein) was incubated with 150 µl of a 50% slurry of IgG–Sepharose (Amersham Pharmacia Biotech) for 4 h. The beads were washed three times with 3 ml of release buffer (20 mM Hepes, pH 7.2, 5 mM MgCl2, 1 mM DTT, 0.75 mM GTP, 0.75 mMGDP, 1 mg/ml BSA) or uptake buffer (20 mM Hepes, pH 7.2, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, 1 mM DTT, 1 mg/ml BSA) and then divided into six equal aliquots. Samples were centrifuged and the supernatant was aspirated. The beads were resuspended in 8 µl of release or uptake buffer and assayed for nucleotide exchange activity. As a control, extract prepared from SFNY823 (MATa ura3-52 L-A-o), which does not contain tagged Bet3p, was incubated with IgG–Sepharose beads in the same way as extract prepared from SFNY904. The beads were processed as described above.
The fold purification of TRAPP was determined by estimating the amount of Trs33p on beads and comparing it to the amount of Trs33p in the lysate. Western blot analysis using the enhanced chemiluminescence method and a standard curve of known amounts of recombinant Trs33p was used to determine the concentration of Trs33p. Previous studies have shown that the components of TRAPP are present in approximately equimolar amounts (Sacher et al. 2000). With this information we were able to calculate the picomoles of TRAPP per mg of protein in the lysate versus the picomoles of TRAPP per mg of protein on the beads. The amount of protein on beads was determined by eluting the washed beads for 5 min at room temperature with 0.2 M glycine (pH 2.8). No protein was detected from untreated beads that were washed in wash buffer and eluted. Protein was measured by the Bradford assay. The method of purification described here results in a preparation that is 12% pure. Comparision of the amount of protein on the beads after incubation with tagged and untagged lysates indicates that the contaminants are not binding in a TRAPP-specific manner.
GDP–GTP Exchange Assay
GDP displacement activity was monitored essentially as described by Jones et al. 1998. Six histidine tagged (His6)-Ypt1p, expressed and purified from E. coli by the protocol of Du et al. 1998, was preloaded by incubating 2 µM of the purified protein with 7.25 µM of [8,5',-3H]GDP (34 Ci/mmol; NEN Life Science Products) in preloading buffer (20 mM Hepes, pH 7.2, 5 mM EDTA, 1 mM DTT) for 15 min at 30°C. At the end of the incubation, MgCl2 was added to a final concentration of 10 mM. The dissociation assay was initiated by the addition of [3H]GDP-Ypt1p (200 nM final concentration) to release buffer in the presence of cell lysates (4 mg/ml) or an aliquot of IgG–Sepharose beads prepared as described above. The reactions (20–30 µl) were performed at 30°C for varying periods of time and terminated by the addition of 1 ml of ice cold stop buffer (20 mM Tris, pH 8.0, 25 mM MgCl2). The reaction mix was filtered on a nitrocellulose filter, washed three times with 3 ml of stop buffer, and the amount of label bound to Ypt1p was measured by scintillation counting (Jones et al. 1998). The protocol for assaying GTP uptake was the same with the following exceptions. Ypt1p at a final concentration of 200 nM was incubated with an aliquot of IgG–Sepharose beads, prepared as described above, and 400 nM of [35S]GTPS (1,000 Ci/mmol; NEN Life Science Products), that was diluted to a specific activity of 25 Ci/mmol. Samples were incubated for the indicated periods of time at room temperature.
The increase in specific exchange activity that resulted as a consequence of the TRAPP purification was determined by estimating the [3H]GDP release activity/mg of protein in the lysate and comparing it to the [3H]GDP release activity/mg of protein on the beads.
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Results |
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S, a nonhydrolyzable analogue of GTP (Ypt1p–GTP). As a control, GST alone was used. The beads were incubated with a lysate prepared from wild-type yeast cells and then probed with antibodies to TRAPP subunits to determine the binding of the different forms of Ypt1p. When Ypt1p was preloaded with either GDP or GTPS (Fig. 1 A, lanes 3 and 4, respectively) small amounts of TRAPP, comparable to background levels (lane 1), bound to beads. However, when Ypt1p was first stripped of nucleotide, binding of TRAPP above background was observed (lane 2). Other tethering factors acting at this stage of the secretory pathway (Uso1p and Sec34p), did not bind to the nucleotide-free form of Ypt1p (data not shown).
To assess the specificity of the interaction between Ypt1p and TRAPP, we examined the interaction of TRAPP with another small GTPase, Ypt51p/Vps21p. Ypt51p/Vps21p mediates membrane traffic on the endosomal and/or vacuolar sorting pathways (Horazdovsky et al. 1994; Singer-Krüger et al. 1994). A GST–Ypt51p fusion protein was expressed in E. coli, purified on glutathione Sepharose beads and stripped of nucleotide. The beads were then incubated with a yeast lysate, washed, and probed with antibodies to TRAPP subunits. As seen in Fig. 1 B, the binding of TRAPP to GST–Ypt51p in its nucleotide-free state was comparable to the GST control (compare lanes 1 and 3), whereas binding to the nucleotide-free form of GST-Ypt1p was much stronger (compare lanes 2 and 3). We estimate that 0.4% of the total amount of TRAPP in the lysate bound to GST-Ypt1p in its nucleotide-free state (Fig. 1 B, compare 0.1% load with lane 2).
If the removal of nucleotide from Ypt1p irreversibly denatures the protein, binding of TRAPP to the nucleotide-free form of Ypt1p could be the result of a nonspecific interaction. To address this possibility, Ypt1p was stripped of nucleotide and then allowed to rebind GDP before it was added to the binding assay. GST–Ypt1p treated in this way showed a greatly reduced ability to bind TRAPP (Fig. 1 B, compare lanes 2 and 4). Thus, the interaction between the nucleotide-free form of Ypt1p and TRAPP is specific and not due to a nonspecific effect of irreversibly denaturating Ypt1p. These results indicate that Ypt1p and TRAPP physically interact with each other, and that TRAPP preferentially binds to the nucleotide-free state of Ypt1p.
An Extract Prepared from the bet3-1 Mutant Fails to Stimulate [3H]GDP Release from Ypt1p
Guanine nucleotide exchange factors (GEF) bind with a higher affinity to the nucleotide-free form of the GTPase than to other forms of the small GTP-binding protein (Lai et al. 1993). The interaction between TRAPP and Ypt1p in its nucleotide-free state suggested that TRAPP may provide guanine nucleotide exchange activity for Ypt1p. To begin to address this possibility, we prepared extracts from various temperature-sensitive (ts) mutants that fail to tether vesicles to the Golgi and then tested their ability to displace GDP from Ypt1p. A mutant that harbors mutations in one of the TRAPP subunits, bet3-1, as well as mutants defective in other components of the tethering machinery, uso1-1, sec34-2, and sec35-1 were examined. Crude cell extracts of wild-type and the mutants were incubated at 30° or 37°C (restrictive temperature) with recombinant Ypt1p preloaded with [3H]GDP. The intrinsic rate of [3H]GDP displacment from Ypt1p was measured in the presence of BSA. Extracts of uso1-1, sec34-2, and sec35-1 stimulated [3H]GDP release from Ypt1p as well as wild type at both 30° and 37°C (Fig. 2a and Fig. b), indicating that exchange activity was not defective in these mutants. In contrast, the bet3-1 mutant was partially defective in the rate of [3H]GDP release from Ypt1p at 30°C (Fig. 2 A) and activity was almost completely abolished at 37°C (Fig. 2 B). These findings imply that TRAPP may displace GDP from Ypt1p.
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S or Ypt1p preloaded with [3H]GDP. Prebound [3H]GDP dissociated from Ypt1p with an intrinsic rate and [35S]GTPS bound with a similar intrinsic rate (Fig. 3 A and B). Purified TRAPP potently stimulated both of these rates when compared with the controls. The uptake of [35S]GTPS in the reaction was dependent on Ypt1p and not other small GTP-binding proteins that may have co-purified with TRAPP (Fig. 3 B, compare TRAPP ± Ypt1p). In addition, the stimulation of nucleotide exchange onto Ypt1p was found to be dependent on the concentration of TRAPP (data not shown). Since small GTPases purified from E. coli are in a complex with GDP (Poe et al. 1985), the uptake of [35S]GTPS onto Ypt1p is a reflection of nucleotide exchange.
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We addressed the substrate specificity of TRAPP, relative to other Rabs, and found that it did not stimulate nucleotide exchange on Ypt32p (Fig. 4 A) and Sec4p (Fig. 4 B). These small GTPases are required for the budding of vesicles from the trans-Golgi and for post-Golgi membrane traffic in yeast, respectively (Salminen and Novick 1987; Benli et al. 1996; Jedd et al. 1997). Thus, guanine nucleotide exchange onto Ypt1p is potently and specifically catalyzed by TRAPP.
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Wild-type and ts mutant extracts were incubated with Ypt1p that was preloaded with [3H]GDP and assayed for release activity at 30° and 37°C as described above. As was found for bet3-1, extracts prepared from the bet5-1, trs31-1, and trs130ts2 mutants were partially defective for activity at 30°C (Fig. 5 A) and more dramatically affected at 37°C (Fig. 5 B). As a control, we also assayed a lysate prepared from trs85. Whereas BET3, BET5, TRS31, and TRS130 are essential for the vegetative growth of yeast, TRS85 is dispensable (Sacher et al. 2000). As anticipated, the absence of Trs85p did not lead to a loss of activity at either 30°C (Fig. 5 A) or 37°C (Fig. 5 B).
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400 ng of purified TRAPP, a minimum of 400 ng of each recombinant subunit was assayed for exchange activity. None of the subunits, however, displayed such an activity (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that TRAPP is the GEF for Ypt1p is consistent with an earlier report that described a specific exchange activity for Ypt1p in a P100 fraction that is enriched for Golgi membranes (Jones et al. 1998). Our findings are also consistent with in vitro transport studies showing that Ypt1p acts on the acceptor compartment (Bacon et al. 1989; Cao and Barlowe 2000). Since Ypt1p is present on ER-derived vesicles but not activated until it encounters TRAPP on the Golgi, we propose it is the GDP-bound, or inactive form, of Ypt1p that resides on this transport intermediate.
Exchange factors for other Rabs have been identified (Horiuchi et al. 1997; Walch-Solimena et al. 1997). Sec2p is the exchange factor for Sec4p, the closest yeast homologue of Ypt1p (Salminen and Novick 1987). The activation of Sec4p by Sec2p is coupled to the polarized delivery of vesicles to the growing bud tip that is the site for exocytosis (Walch-Solimena et al. 1997). Rabex-5 is the GEF for Rab5. It forms a complex with the Rab5 effector, Rabaptin-5. The Rabex-5/Rabaptin-5 complex is essential for endocytic membrane fusion (Horiuchi et al. 1997). Sec2p, Rabex 5, and its yeast homologue Vps9p (Hama et al. 1999), however, do not share homology with any of the 10 TRAPP subunits.
ts mutants in 4 of the 10 genes that encode TRAPP subunits (bet3-1, bet5-1, trs31-1, and trs130ts2) have been isolated (Rossi et al. 1995; Jiang et al. 1998; Sacher, M., manuscript in preparation). Extracts prepared from these mutants displayed ts defects in their ability to stimulate the release of GDP from Ypt1p. Some of these mutations disrupt the complex (bet3-1 and bet5-1), while others do not (trs130ts2) (Barrowman, J., and S. Ferro-Novick, unpublished observations). Thus, the conformation of TRAPP may be important for stimulating nucleotide exchange on Ypt1p. Consistent with this hypothesis, we have found that purified recombinant forms of each of the TRAPP subunits do not accelerate the release of GDP from Ypt1p or stimulate the uptake of GTP. Alternatively, one or more subunits may be required to stabilize the component(s) of the complex that has exchange activity. The observation that exchange activity is almost completely abolished in the bet3-1 and trs31-1 mutants implies that TRAPP is the major exchange factor for Ypt1p.
Previous findings have shown that a dominant mutation in SLY1 (SLY1-20), which encodes a soluble NSF attachment protein receptor–associated protein, can bypass the loss of YPT1 (Dascher et al. 1991). The finding that the loss of TRAPP cannot be bypassed by SLY1-20 (Barrowman et al. 2000) implies that, in addition to stimulating nucleotide exchange on Ypt1p, TRAPP has other roles. Nonetheless, the identification of TRAPP as the GEF for Ypt1p has begun to shed light on the molecular mechanism by which TRAPP tethers/targets vesicles to the Golgi.
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Acknowledgments |
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mutants, Birgit Singer-Krüger for the YPT-GST expression vectors, N. Barry Elkind for technical advice on nucleotide exchange assays, Li-Lin Du and Chave Carr for a critical reading of the manuscript, and Joyce Anquillare for assistance in the preparation of this manuscript. W. Wang is supported as an Associate of the Howard Hughes Medical Institute.
Submitted: 5 April 2000
Revised: 6 September 2000
Accepted: 8 September 2000
Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GST, glutathione-S-transferase; His6, six histidine tag; ts, temperature-sensitive.
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