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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 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).
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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).

Mentions: 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.


TRAPP stimulates guanine nucleotide exchange on Ypt1p.

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

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).
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Related In: Results  -  Collection

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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).
Mentions: 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.

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