Study on Protein Secretion of Yeast by a Temperature-sensitive Mutant

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The uso1 mutant of S. cerevisiae has a defect in the transport of protein from the ER to the Golgi. We cloned the wild-type USO1 gene and determined the nucleotide sequence of 5937 bp. The USO1 gene was found to be essential for growth by gene disruption experiments and was located on the chromosome IV by pulse-field gel electrophoresis. The longest open reading frame starts at ATG (273) and ends at TGA (5607) and codes a protein of 1790 amino acids. This protein is hydrophilic as a whole without any long hydrophobic stretches and has two acidic stretches at 468th and 1771st amino acid. The former acidic stretch contains a calcium-binding motif. The C-terminal half of the protein should form a coiled-coil alpha-helix and the protein should exist as a dimer. We constructed hybrid genes containing codons 36-571 or 1128-1790 of Usolp and expressed them in E. coli. After purifying the fusion roteins, we immunized mice and rabbits with them and obtained anti-Usolp antiserums. The Usolp protein was detected by the rabbit antiserum when USO1 was highly expressed by the GAL7 promoter but not in the wild-type yeasts or yeasts with multicopy USO1 gene. As the mRNA of USO1 was also detected by Nothern blots only when USO1 was expressed by the GAL7 promoter, very small amount of Uso1p should exist and function in yeast cell.