Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells

Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the ce ll surface remain obscure. We describe here a mutant able to export enhance d amount of proteins, Classical genetic methods, nucleic acids manipulation s and cloning procedures were used to isolate and characterize the mutant a nd to clone and sequence the corresponding wild type gene. The isolated Sac charomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type s train (80 mu g of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 mu g of proteins/1 x 10(8) parental cells, SEM +/- I, n22). Protein overex port in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene ide ntified an open reading frame of 786 bp coding for a hydrophilic protein wi th predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It i s concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisi ae cells, (C) 1999 Elsevier Science Ltd. All rights reserved.