Analysis of alpha-factor secretion signals by fusing with acid phosphatase of yeast.
In yeast, Saccharomyces cerevisiae, the PHO5 gene encodes the repressible acid phosphatase (APase) whose activity can be easily monitored by either the staining of colonies or by colorimetric assay. Therefore, gene fusions to PHO5 provide a convenient system for structural and functional analysis of yeast genes. We have constructed fusions of the PHO5 gene with a MF alpha 1 gene of yeast to delineate the secretion signal(s) in the alpha-factor leader peptide. Gene fusion between MF alpha 1 and PHO5 codes for a hybrid protein in which the alpha-factor leader peptide of 89 amino acids (aa) directed the export of APase, a periplasmic protein, into the medium. Since the hybrid gene is transcribed from the alpha-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation. Further analyses of another MF alpha 1-PHO5 fusion showed that only the first 22 aa of the 89-aa alpha-factor leader peptide contained sufficient information for the secretion of APase into the medium. This shows that, in addition to the analysis of gene regulation, PHO5 fusions can be used to study signals involved in the proper localization of proteins.
Sidhu
R S
RS
Department of Molecular Genetics, Wadley Institutes of Molecular Medicine, Dallas, TX 75235.
Bollon
A P
AP
D000135:Acid Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005800:Genes, Fungal; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D010455:Peptides; D000072235:Mating Factor; D004262:DNA Restriction Enzymes; D000135:Acid Phosphatase