The yeast homolog of H < beta > 58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole.
The highly conserved and ubiquitously expressed mouse gene H < beta > 58, identified through insertional mutagenesis, has been shown to be essential for early postimplantation development in mouse, but the mechanism by which it acts is unknown (Radice et al. 1991; Lee et al. 1992). We report here the identification of a yeast gene related to the H < beta > 58 gene and provide biochemical and genetic evidence for its function within the cell. The gene, PEP8, plays a role in the delivery of proteins to the vacuole. Disruption of the gene did not affect cell viability. However, the disruptants were shown to have a defect in the processing of the soluble vacuolar proteases but not the membrane vacuolar hydrolases. The processing defect appeared to be a consequence of the inability of the soluble vacuolar hydrolase to reach the vacuole. Although a small amount of the vacuolar precursors was mis-sorted to the extracellular medium, mis-sorting did not appear to be the primary defect in these cells. Pep8p was identified by epitope tagging of the protein. Biochemical fractionation indicated that the protein was peripherally bound to membranes. Immuno-gold electron microscopy indicated that the Pep8p localized to vacuolar membranes. Complementation experiments with the mouse H < beta > 58 cDNA revealed that a Pep8p-H < beta > 58 fusion protein in which the carboxy-terminal 85 amino acids of Pep8p were replaced by the carboxy-terminal 115 amino acids of H < beta > 58 was functional.(ABSTRACT TRUNCATED AT 250 WORDS)
Bachhawat
A K
AK
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Suhan
J
J
Jones
E W
EW
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002451:Cell Compartmentation; D004252:DNA Mutational Analysis; D010064:Embryo Implantation / Q000235:genetics; D005260:Female; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics*; D051379:Mice / Q000196:embryology; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; C087335:PEP1 protein, S cerevisiae; C089990:PEP8 protein, S cerevisiae; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C089989:Vps26 protein, mouse; D000469:Alkaline Phosphatase