A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small cytosolic factor which inhibits vacuole inheritance.
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 Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l 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.
Nicolson
T
T
Molecular Biology Institute and Department of Biological Chemistry, University of Los Angeles, California 90024, USA.
Conradt
B
B
Wickner
W
W
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002474:Cell-Free System; D016213:Cyclins / Q000235:genetics*; D003600:Cytosol / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects; D008954:Models, Biological; D010446:Peptide Fragments / Q000235:genetics; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000235:genetics*
D016213:Cyclins; D005656:Fungal Proteins; C066824:PHO80 protein, S cerevisiae; D010446:Peptide Fragments; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate