TRAPP stimulates guanine nucleotide exchange on Ypt1p - PubMed (original) (raw)
TRAPP stimulates guanine nucleotide exchange on Ypt1p
W Wang et al. J Cell Biol. 2000.
Abstract
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.
Figures
Figure 1
TRAPP subunits bind preferentially to the nucleotide-free form of Ypt1p. (A) A yeast lysate was incubated with agarose beads containing either GST (lane 1) or GST-Ypt1p (lanes 2–4) as described in the Materials and Methods. Before the incubation, Ypt1p was either stripped of nucleotide (nucleotide-free, lane 2) or loaded with GDP (lane 3) or GTPγS (lane 4). The beads were washed and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. The eluate was then fractionated on a SDS–12.5% polyacrylamide gel, and Western blot analysis was performed by the enhanced chemiluminescence method using anti-Trs33p antibody at 1:2,500 dilution (top) or anti-Trs20p antibody at 1:1,000 dilution (bottom). (B) A yeast lysate was incubated with agarose beads containing either GST (lane 1), GST-Ypt1p (lanes 2 and 4), or GST-Ypt51p (lane 3). Before the incubation, Ypt1p and Ypt51p were stripped of nucleotide (nucleotide-free, lanes 2 and 3) or stripped of nucleotide and allowed to rebind GDP (lane 4). The beads were processed as above. The amount of Trs33p present in 0.1% of the lysate that was incubated with the beads is shown.
Figure 1
TRAPP subunits bind preferentially to the nucleotide-free form of Ypt1p. (A) A yeast lysate was incubated with agarose beads containing either GST (lane 1) or GST-Ypt1p (lanes 2–4) as described in the Materials and Methods. Before the incubation, Ypt1p was either stripped of nucleotide (nucleotide-free, lane 2) or loaded with GDP (lane 3) or GTPγS (lane 4). The beads were washed and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. The eluate was then fractionated on a SDS–12.5% polyacrylamide gel, and Western blot analysis was performed by the enhanced chemiluminescence method using anti-Trs33p antibody at 1:2,500 dilution (top) or anti-Trs20p antibody at 1:1,000 dilution (bottom). (B) A yeast lysate was incubated with agarose beads containing either GST (lane 1), GST-Ypt1p (lanes 2 and 4), or GST-Ypt51p (lane 3). Before the incubation, Ypt1p and Ypt51p were stripped of nucleotide (nucleotide-free, lanes 2 and 3) or stripped of nucleotide and allowed to rebind GDP (lane 4). The beads were processed as above. The amount of Trs33p present in 0.1% of the lysate that was incubated with the beads is shown.
Figure 2
[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that block vesicle tethering. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The amount of [3H]GDP bound to His6-Ypt1p was measured using a filter binding assay (Jones et al. 1998). The data are expressed as the percent of radioactivity released from Ypt1p relative to the amount bound before the incubation. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (13.0 ± 2% at 30°C; 26.7 ± 4% at 37°C) was subtracted as background. The numbers reported are the average of three separate experiments.
Figure 2
[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that block vesicle tethering. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The amount of [3H]GDP bound to His6-Ypt1p was measured using a filter binding assay (Jones et al. 1998). The data are expressed as the percent of radioactivity released from Ypt1p relative to the amount bound before the incubation. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (13.0 ± 2% at 30°C; 26.7 ± 4% at 37°C) was subtracted as background. The numbers reported are the average of three separate experiments.
Figure 3
TRAPP stimulates guanine nucleotide exchange on Ypt1p. (A) To measure [3H]GDP dissociation from Ypt1p, His6-Ypt1p was preloaded with [3H]GDP and then incubated for various times at 30°C with IgG–Sepharose beads containing TRAPP or lacking it (see Control). The beads were prepared as described in the Materials and Methods. For each time point, the data are expressed as the percent of label bound to Ypt1p compared with the zero time point. (B) To measure the uptake of [35S]GTPγS onto Ypt1p, IgG–Sepharose beads with TRAPP or without it (see control) were incubated at room temperature in the absence or presence of His6-Ypt1p plus [35S]GTPγS. Radioactivity bound to Ypt1p was measured at the indicated periods of time.
Figure 3
TRAPP stimulates guanine nucleotide exchange on Ypt1p. (A) To measure [3H]GDP dissociation from Ypt1p, His6-Ypt1p was preloaded with [3H]GDP and then incubated for various times at 30°C with IgG–Sepharose beads containing TRAPP or lacking it (see Control). The beads were prepared as described in the Materials and Methods. For each time point, the data are expressed as the percent of label bound to Ypt1p compared with the zero time point. (B) To measure the uptake of [35S]GTPγS onto Ypt1p, IgG–Sepharose beads with TRAPP or without it (see control) were incubated at room temperature in the absence or presence of His6-Ypt1p plus [35S]GTPγS. Radioactivity bound to Ypt1p was measured at the indicated periods of time.
Figure 4
TRAPP does not stimulate the uptake of [35S]GTPγS onto Ypt32p or Sec4p. Equal amounts of His6-Ypt32p (A), or His6-Sec4p (B) and His6-Ypt1p were incubated with [35S]GTPγS at room temperature with IgG–Sepharose beads, containing TRAPP or lacking it (see Control) exactly as described above for His6-Ypt1p. At the indicated periods of time, the radioactivity bound to protein was measured. Note that Sec4p has a high intrinsic exchange rate as previously reported (Kabcenell et al. 1990).
Figure 4
TRAPP does not stimulate the uptake of [35S]GTPγS onto Ypt32p or Sec4p. Equal amounts of His6-Ypt32p (A), or His6-Sec4p (B) and His6-Ypt1p were incubated with [35S]GTPγS at room temperature with IgG–Sepharose beads, containing TRAPP or lacking it (see Control) exactly as described above for His6-Ypt1p. At the indicated periods of time, the radioactivity bound to protein was measured. Note that Sec4p has a high intrinsic exchange rate as previously reported (Kabcenell et al. 1990).
Figure 5
[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.
Figure 5
[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.
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