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We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase activating proteins family (GAP): Gyp5p, a potent GAP in vitro for Ypt1p and Sec4p, and the protein Ymr192wp/APP2 that we propose to rename Gyl1p (GYp like protein). Immunofluorescence experiments showed that Gyp5p and Gyl1p partly colocalize at the bud emergence site, at the bud tip and at the bud neck during cytokinesis. Subcellular fractionation and co-immunoprecipitation experiments showed that Gyp5p and Gyl1p co-fractionate with post-Golgi vesicles and plasma membrane, and belong to the same protein complexes in both localizations. We found by co-immunoprecipitation experiments that a fraction of Gyp5p interacts with Sec4p, a small GTPase involved in exocytosis, and that a fraction of Gyl1p associates at the plasma membrane with the Gyp5p/Sec4p complexes. We showed also that GYP5 genetically interacts with SEC2, which encodes the Sec4p exchange factor. Examination of the gyp5Δgyl1Δ mutants grown at 13°C revealed a slight growth defect, a secretion defect and an accumulation of secretory vesicles in the small-budded cells. These data suggest that Gyp5p and Gyl1p are involved in control of polarized exocytosis.

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Isolated nuclei, prepared from myeloblasts of chicks infected with avian myeloblastosis virus, synthesize RNA sequences present in avian myeloblastosis viral RNA. These sequences are also formed during transcription of chromatin, isolated from myeloblasts, by DNA-dependent RNA polymerases purified from Escherichia coli or calf thymus. In the latter case, transcription is a-amanitin sensitive. Formation of hybrids between RNA and avian myeloblastosis virus DNA probes has been monitored by the combined use of ribonucleases A, T1, and H, and ribonucleases specific for single strands.