Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor.
We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.
Young
M R
MR
Department of Biological Chemistry, University of Maryland, Baltimore 21201.
Andreadis
J
J
Hu
L T
LT
Wolfe
P B
PB
D000135:Acid Phosphatase / Q000235:genetics / Q000502:physiology*; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism*; D043402:Cathepsin A; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000502:physiology; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D000072235:Mating Factor; D008861:Microsomes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000502:physiology*
D006360:Heat-Shock Proteins; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D000135:Acid Phosphatase; D001616:beta-Galactosidase; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase