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TRAPP stimulates guanine nucleotide exchange on Ypt1p.

Wang W, Sacher M, Ferro-Novick S - J. Cell Biol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Cell Biology Yale University School of Medicine, New Haven, Connecticut 06519-1418, USA.

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.

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[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.
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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.

Mentions: 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.


TRAPP stimulates guanine nucleotide exchange on Ypt1p.

Wang W, Sacher M, Ferro-Novick S - J. Cell Biol. (2000)

[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.
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Related In: Results  -  Collection

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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.
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Cell Biology Yale University School of Medicine, New Haven, Connecticut 06519-1418, USA.

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.

Show MeSH
Related in: MedlinePlus