Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes.

Abstract

Abstract. Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pHdependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl-mutants are defective in endocytosis. Four of these mutant strains (endS-l, end9-1, endlO-l, and end11-1 ) show altered uptake of the endocytosed ligand, a-factor, and three

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

“…An additional test was performed to specifically identify disruptants impaired in vacuolar targeting via the CPY pathway. This test was based on colony immunoblotting to detect the secretion of CPY, a sorting defect exhibited by a subset of endocytic (end) and vacuolar protein sorting (vps) mutants (Munn and Riezman, 1994;Roberts et al, 1991). We identified 25 mutants among the 631 viable deletion strains included in our screens, that correspond to unknown genes or genes recently identified as being involved in trafficking or glycosylation.…”

“…The filter was then removed from the plate, the cells washed off with water and we tested for immunodetectable CPY, as described above (Roberts et al, 1991). All the tests included the following controls: both wild-type strains, and two mutant strains, vps1 (Rothman et al, 1990), defective in a dynamin-like GTPase and vps4/end13 (Babst et al, 1998;Munn and Riezman, 1994;Zahn et al, 2001), defective in an AAA-ATPase, both proteins required for vacuolar protein sorting and both mutants displaying CPY secretion (Munn and Riezman, 1994).…”

“…CPY is finally transported via late endosomes to the vacuole, where it is processed to give the 61 kDa mature species (mCPY) by proteinase A (PrA), the product of the PEP4 gene (Ammerer et al, 1986). Many vps mutants generally accumulate p2 CPY, which is secreted to the medium where it is detected by colony immunoblotting (Munn and Riezman, 1994). ALP has an internal transmembrane segment but no signal peptide.…”

A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy

“…Isolation of a P. pastoris homologue of VPS15 Previously, we cloned the H. polymorpha PDD1 gene (Kiel et al 1999) and discovered that it is highly homologous to S. cerevisiae VPS34, a gene required for vacuolar protein sorting (Herman and Emr 1990) and endocytosis (Munn and Riezman 1994). In S. cerevisiae, ScVps34p function has been shown to be dependent on activation by the protein kinase ScVps15p (Stack et al 1995 a,b).…”

“…Later, Scvps34 mutants were independently found among mutants defective in endocytosis (end12 mutants; Munn and Riezman 1994), indicating that sorting of proteases to the vacuole and late steps in endocytosis require at least some common gene products (Munn and Riezman 1994). Disruption of either ScVPS34 or ScVPS15 results in a set of speci®c phenotypes including temperature-sensitive growth, defects in osmoregulation, disturbed vacuolar segregation, and missorting of multiple soluble vacuolar hydrolases.…”