Fermentation Technology, Hiroshima University, 1-4-1 Higas h i-H iros h i ma 739, Japan 84322-5305, USAThe Preudomnas syringae cyclic lipodepsipeptide syringomycin inhibits the growth of Saccharomyces cemwisi4e. A novel yeast gene, SYR2, was found to complement two syringomycin-resistant S. cemwisiae mutants. SYR2 was cloned, sequenced, and shown to encode a 349 amino acid protein located in the endoplasmic reticulum. SYR2 was identical to SUR2, which is involved in survival during nutritional starvation. Gene disruption or overexpression of SYR2 did not affect cell viability or ergosterol levels, but did influence cellular phospholipid levels. The findings suggest that phospholipids are important for the growth inhibitory action of syringomycin.

A 2.5 kb DNA fragment of the Saccharomyces cerevisiae S Y . 7 gene was cloned by complementation of the syrl mutations that simultaneously lead t o 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. SYR7 was identical to ERG3, which is suggested to encode C-5 sterol desaturase required for ergosterol biosynthesis. The protein product of SYR7 was identified by Western blot analysis as a protein of 40 kDa in the particulate fraction. Gene disruption experiments demonstrated that elimination of SYR7IERG3 is not lethal, but results in membrane C-5 desaturated sterol deficiencies, resistance to syringomycin and sensitivity to high Ca2+. The syrl 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.

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H+-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H'-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H -ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca2". Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H+-ATPase and that specific protein kinase(s) are probably associated with the enzyme. SR2 is a virulence factor produced by certain plant pathogenic strains of Pseudomonas syringae pv syringae (30). This 1226-D lipodepsipeptide (9, 28) affects the plasma membranes of plants and yeast (30). SR alters the charge potential and Ca2" and K+ fluxes across these plasma membranes (15,25,31,34). The closure of stomata that it induces in whole leaves of Vicia faba (21) (3,34). It also stimulates the