During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.
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| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| VAC7 | growth in exponential phase: decreased | classical genetics | reduction of function Allele: vac7-1 | SEY6210 | Details: mutant cells have a doubling time of 2.5 hours in rich medium at 30°C compared to 1.6 hours for wt cells | |
| VAC7 | vacuolar distribution: abnormal | classical genetics | reduction of function Allele: vac7-1 | SEY6210 | Details: vacuolar segregation defect; 26% of cells contain a vacuole that traverses mother and daughter cells with a gap at the neck of the junction (an open figure eight); indicative of vacuoles that fails to undergo membrane scission | |
| FAB1 | vacuolar distribution: abnormal | classical genetics | conditional Allele: fab1-2 G864E; CCT domain mutated; disrupts interaction with Vac14p | SEY6210 | Temperature: elevated temperature, 37 °C Details: vacuolar segregation defect; high percentage of cells contain a vacuole that traverses mother and daughter cells with a gap at the neck of the junction; indicative of vacuoles that fails to undergo membrane scission | |
| VAC14 | vacuolar distribution: abnormal | classical genetics | conditional Allele: vac14-1 | SEY6210 | Temperature: elevated temperature, 37 °C Details: vacuolar segregation defect; high percentage of cells contain a vacuole that traverses mother and daughter cells with a gap at the neck of the junction; indicative of vacuoles that fails to undergo membrane scission | |
| VAC7 | vacuolar morphology: abnormal | classical genetics | reduction of function Allele: vac7-1 | SEY6210 | Details: class III mutant cells contain dramatically enlarged (swollen), unlobed vacuoles in contrast to wt cells that contain multilobed vacuoles of smaller size; vacuole acidification defect | |
| FAB1 | vacuolar morphology: abnormal | classical genetics | conditional Allele: fab1-2 G864E; CCT domain mutated; disrupts interaction with Vac14p | SEY6210 | Temperature: elevated temperature, 37 °C Details: class III mutant cells contain dramatically enlarged (swollen), unlobed vacuoles at elevated temperature in contrast to wt cells that contain multilobed vacuoles of smaller size; vacuole acidification defect | |
| VAC14 | vacuolar morphology: abnormal | classical genetics | conditional Allele: vac14-1 | SEY6210 | Temperature: elevated temperature, 37 °C Details: class III mutant cells contain dramatically enlarged (swollen), unlobed vacuoles in contrast to wt cells that contain multilobed vacuoles of smaller size; vacuole acidification defect |