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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 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.
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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.

Mentions: 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 GTPγS 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.


TRAPP stimulates guanine nucleotide exchange on Ypt1p.

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

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.
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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.
Mentions: 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 GTPγS 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.

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