IDENTIFICATION AND ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE SYR1 GENE REVEALS THAT ERGOSTEROL IS INVOLVED IN THE ACTION OF SYRINGOMYCIN

A 2.5 kb DNA fragment of the Saccharomyces cerevisiae SYR1 gene was cloned by complementation of the syr1 mutations that simultaneously lead to resistance to the phytotoxin syringomycin and sensitivity of growth to high Ca2+ concentrations. Sequencing of this fragment revealed a single open reading frame encoding a polypeptide of 365 amino acids. Four hydrophobic regions each separated by hydrophilic regions were present in the protein. SYR1 was identical to ERG3, which is suggested to encode C-5 sterol desaturase required for ergosterol biosynthesis. The protein product of SYR1 was identified by Western blot analysis as a protein of 40 kDa in the particulate fraction. Gene disruption experiments demonstrated that elimination of SYR1/ERG3 is not lethal, but results in membrane C-5 desaturated sterol deficiencies, resistance to syringomycin and sensitivity to high Ca2+. The syr1 mutant cells had significantly decreased ability for syringomycin binding. The results indicated that C-5 desaturated sterols are involved in the binding of syringomycin to the cell, and the lack of the sterols in the mutant membrane results in sensitivity to high Ca2+ and an increased rate of cellular Ca2+ influx.