Table 2.

Activity of Ypt1p regulators in extracts of mutant strains

A	Yeast strain	% GDP release
	DSS4 wild-type (NY 931)	40
	Δdss4-(NY929)	42
	Wild-type (GPY60)	39
B	Yeast strain	% GAP activity in the P12
	Wild type (NSY116)	100
	ΔGYP1 ΔGYP7 (NSY420)	93.3
C	Yeast strain	GAP activity in the P12
	Wild type (NY13)	+
	sec22-3 (NY426)	+
	sec18-1 (NY431)	+
	sec17-1 (NY418)	+

(A) The Ypt1p-GEF is not Dss4p. [³H]GDP release assays were performed in the presence of 1 mg/ml P100 fraction prepared from strains carrying either a dss4 deletion (NY929) or the wild-type DSS4 allele (NY931). Both strains yielded equivalent exchange activities, similar to the activity observed with the wild-type strain (GPY60) used for this study. Results show percent stimulated GDP release after 15 min of incubation and are representative of two experiments with results agreeing to within 5%. For comparison, <5% of the bound GDP was released after a 15-min incubation in the absence of cell extract. (B) Deletion of GYP1 and GYP7 does not affect the amount of GAP activity in the P12. Two nanomolar Ypt1p preloaded with [γ-³²P]GTP was incubated at 30°C for 30 min with 0.5 mg/ml P12 generated from fractionation of yeast strains containing either the wild-type alleles of GYP1 and GYP7 or deletions of these genes. BSA (1 mg/ml) was included in the measurement of intrinsic hydrolysis. GTP hydrolysis was measured by the charcoal assay, and results are expressed as percent of the GAP activity in wild-type P12. Data shown are an average of two independent experiments, with results agreeing to within 5%. (C) The Ypt1p-GAP activity is not influenced by mutations in genes whose products are involved in vesicle fusion: SEC18, SEC17, or SEC22. GAP assays were performed as above, using the TLC method. Both the P12 and the S12 fractions were tested. Mutant cells showed the same distributions and levels of Ypt1p-GAP as wild-type cells.