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J Biol Chem

Title:		Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor
Author(s):		Young MR; Andreadis J; Hu LT; Wolfe PB;
Address:		"Department of Biological Chemistry, University of Maryland, Baltimore 21201"
Journal Title:		J Biol Chem
Year:		1990
Volume:		265
Issue:		32
Page Number:		19824 - 19832
DOI:
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"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"
Keywords:		Acid Phosphatase/genetics/*physiology Biological Transport Carboxypeptidases/*metabolism Cathepsin A Endoplasmic Reticulum/metabolism Fluorescent Antibody Technique Gene Expression Heat-Shock Proteins/physiology Immunoblotting Intracellular Membranes/meta;
Notes:		"MedlineYoung, M R Andreadis, J Hu, L T Wolfe, P B eng GM35145/GM/NIGMS NIH HHS/ Research Support, U.S. Gov't, P.H.S. 1990/11/15 J Biol Chem. 1990 Nov 15; 265(32):19824-32"