Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: Ypt1p-Q67L is defective in intrinsic and GAP-stimulated GTP hydrolysis. GTP hydrolysis was monitored by the charcoal binding assay. Wild-type (squares) and Ypt1p-Q67L (triangles) proteins were preloaded with [γ-32P]GTP for 15 min at 30°C. Unbound nucleotide was removed with two successive acrylamide spin columns. GTP hydrolysis assays were performed by incubating 2 nM preloaded Ypt1p with a P12 subcellular fraction (5 mg/ml) prepared from GPY60 cells (GAP-stimulated hydrolysis; open symbols) or without the P12 fraction (intrinsic hydrolysis; closed symbols) at 30°C. Aliquots were removed at the indicated time points and added to ice-cold activated charcoal to stop the reaction. The charcoal was pelleted, and an aliquot of the supernatant was removed and quantified by scintillation counting. The counts measured at time zero were subtracted as background. GTP binding was slightly less efficient for Ypt1p-Q67L, but hydrolysis rates were normalized for the amount of Ypt1p bound to GTP. Data shown are typical of three independent experiments.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Binding Assay

Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: Mutant Ypt1p-Q67L is partially defective in prenylation. (A) A smaller fraction of the mutant Ypt1p-Q67L than of wild-type Ypt1p is prenylated, as determined by Triton X-114 phase partitioning and urea-acrylamide gradient gel electrophoresis. Wild-type (NSY125) and ypt1-Q67L mutant (NSY406) total cell lysates were subjected to phase partitioning with 1% Triton X-114. Total (T), aqueous (A), and detergent (D) phases were electrophoresed on 4 to 8 M urea–10 to 15% acrylamide gels and processed for Western blot analysis with anti-Ypt1p antibodies. Note that the aqueous phase contains all of the unprenylated form and the detergent phase contains all of the prenylated Ypt1p-Q67L. (B) Unprenylated and some prenylated mutant Ypt1p-Q67L is mislocalized to the cytoplasm (S100 fraction). Wild-type and ypt1-Q67L mutant cells were lysed with glass beads and centrifuged at 100,000 × g to generate supernatant (S) and pellet (P) fractions (T, total cell lysate). Proteins were resolved by SDS-PAGE on 4 to 8 M urea–10 to 15% acrylamide gradient gels, transferred to nylon membranes, and processed for Western blot analysis with affinity-purified anti-Ypt1p antibodies. The upper form of Ypt1p is unprenylated; the lower form is prenylated. Quantification indicates that there is the same amount of prenylated Ypt1p in wild-type and mutant strains. Data are typical of three independent experiments.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Mutagenesis, Nucleic Acid Electrophoresis, Western Blot, SDS Page, Affinity Purification

Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: ypt1-Q67L is not dominant for growth or secretion when overexpressed. (A) Growth phenotypes. Wild-type YPT1 (WT; pNS326), ypt1-Q67L (pNS330), and YPT1-N121I (pNS327) were expressed from the galactose-inducible GAL10 promoter on a CEN URA-marked plasmid in the strain NSY125. Tenfold serial dilutions of cells were spotted onto SRaf-Ura or SRaf-Ura-plus-2% galactose plates and grown at 30°C. (B) CPY transport. The strains were grown overnight in SRaf-Ura minus methionine at 30°C to mid-logarithmic phase and then switched to inducing media (SRaf-Ura minus methionine plus 2% galactose) for 3 h at 30°C. Cells were harvested and pulse-labeled with Tran35S-label for 6 min at 30°C and chased for the indicated times (minutes). Cells were then lysed, and CPY was immunoprecipitated with anti-CPY antibodies and separated on SDS–8% polyacrylamide gels. p1, ER form; p2, Golgi form; m, mature vacuolar form. (C) Western blot analysis of Ypt1p expression. Cells used for the CPY assay were tested for Ypt1p expression. Ypt1p expression was induced with 2% galactose for 3 h at 30°C. Cells were lysed, and equivalent quantities of extract were run on 4 to 8 M urea–10 to 15% acrylamide SDS-containing gels. Proteins were transferred to nylon membranes and processed for Western blotting with anti-Ypt1p antibodies. The unprenylated and prenylated forms of Ypt1p are indicated.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Plasmid Preparation, Labeling, Immunoprecipitation, Western Blot, Expressing