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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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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.
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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.

Mentions: If TRAPP is an exchange factor for Ypt1p, it should stimulate the uptake of GTP onto Ypt1p as well as displace GDP. Because we were unable to measure GTP uptake in yeast extracts, we could not assess if bet3-1 was defective for this activity. Therefore, we purified the TRAPP complex and measured the ability of the purified complex to stimulate GTP uptake by Ypt1p. A strain (SFNY904; Sacher et al. 2000) in which the sole copy of Bet3p was fused at its carboxy terminus to protein A was used for the purification. Lysates prepared from SFNY904, as well as a strain that does not contain the fusion protein (SFNY823), were treated with IgG–Sepharose as described in the Materials and Methods. The beads were washed and incubated with Ypt1p and [35S]GTPγS or Ypt1p preloaded with [3H]GDP. Prebound [3H]GDP dissociated from Ypt1p with an intrinsic rate and [35S]GTPγS 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]GTPγS 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]GTPγS onto Ypt1p is a reflection of nucleotide exchange.


TRAPP stimulates guanine nucleotide exchange on Ypt1p.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192651&req=5

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.
Mentions: If TRAPP is an exchange factor for Ypt1p, it should stimulate the uptake of GTP onto Ypt1p as well as displace GDP. Because we were unable to measure GTP uptake in yeast extracts, we could not assess if bet3-1 was defective for this activity. Therefore, we purified the TRAPP complex and measured the ability of the purified complex to stimulate GTP uptake by Ypt1p. A strain (SFNY904; Sacher et al. 2000) in which the sole copy of Bet3p was fused at its carboxy terminus to protein A was used for the purification. Lysates prepared from SFNY904, as well as a strain that does not contain the fusion protein (SFNY823), were treated with IgG–Sepharose as described in the Materials and Methods. The beads were washed and incubated with Ypt1p and [35S]GTPγS or Ypt1p preloaded with [3H]GDP. Prebound [3H]GDP dissociated from Ypt1p with an intrinsic rate and [35S]GTPγS 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]GTPγS 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]GTPγS onto Ypt1p is a reflection of nucleotide exchange.

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