Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.
pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.
Woolford
C A
CA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Dixon
C K
CK
Manolson
M F
MF
Wright
R
R
Jones
E W
EW
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006867:Hydrolases / Q000378:metabolism; D015151:Immunoblotting; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D003062:Codon; D005656:Fungal Proteins; C065710:PEP5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006867:Hydrolases; D000469:Alkaline Phosphatase