Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle. vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of wild-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein fraction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-1 small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.
ASJC Scopus subject areas
- Molecular Biology