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.

• Publication year

  1990

• Venue

  Journal of Biological Chemistry

• Publication date

  1990-11-15

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(17)45447-0PMID 2123190
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Purification of the Escherichia coli secB gene product and demonstration of its activity in an in vitro protein translocation system.

  1989cited by this paper
• 70K heat shock related proteins stimulate protein translocation into microsomes

  1988cited by this paper
• PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae.

  1988cited by this paper
• ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

  1988cited by this paper
• The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein.

  1988cited by this paper
• A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides

  1988cited by this paper
• The ATP requiring step in assembly of M13 procoat protein into microsomes is related to preservation of transport competence of the precursor protein.

  1987cited by this paper
• Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form.

  1987cited by this paper
• Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae

  1987cited by this paper
• Cell biology: An unfolding story of protein translocation

  1986cited by this paper
• Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y

  1986cited by this paper
• In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor.

  1986cited by this paper
• A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae

  1986cited by this paper
• Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor

  1986cited by this paper
• Disulfide bonds and the translocation of proteins across membranes.

  1986cited by this paper
• Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria

  1986cited by this paper
• Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae

  1984cited by this paper
• Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor.

  1982cited by this paper
• Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.

  1982cited by this paper
• Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

  1979cited by this paper
• The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

  1979cited by this paper
• Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase.

  1977cited by this paper
• Biosynthesis of acid phosphatase of baker's yeast. Factors influencing its production by protoplasts and characterization of the secreted enzyme.

  1972cited by this paper

• Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery.

  1997cites this paper
• Molecular chaperones and intracellular protein translocation.

  1995cites this paper
• Overexpression of the ER-membrane protein P-450 CYP52A3 mimics sec mutant characteristics in Saccharomyces cerevisiae.

  1993cites this paper
• Protein‐specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

  1992cites this paper