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. 2000 Oct 16;151(2):289–296. doi: 10.1083/jcb.151.2.289

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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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.

[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.