An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast.

Abstract

The peptide mating pheromone .alpha.-factor and the hydrolytic enzyme invertase (.beta.-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (S. cerevisiae). An in-frame fusion of the structural genes for these 2 proteins was constructed by connecting the 5''-flanking region and preproleader portion of the coding sequence of the .alpha.-factor gene (MF.alpha.1) to a large fragment of the invertase gene (SUC2) lacking its 5''-flanking region and the coding information for the first 4 amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the .alpha.-factor precursor were retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-.alpha.-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the .alpha.-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF.alpha.1, the MF.alpha.1-SUC2 fusion is expressed in .alpha. haploids at levels 65-75 times higher than in a haploids or in a/.alpha. diploids; also, high-level expression is eliminated in mat.alpha.1 mutants but not in mat.alpha.2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5''-flanking region present in the fusion (about 950 base pairs) is sufficient to confer .alpha. cell-specific expression to the hybrid gene.