Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H⁺-ATPase from Red Beet Storage Tissue

Abstract

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

Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation)

“…Changes in membrane protein phosphorylation states by nonspecific elicitors have been reported in soybean (Grab et al, 1989), tomato (Felix et al, 1991), and parsley cells (Renelt et al, 1993). Also, treatment of red beet cells with the bacterial toxin, syringomycin, resulted in phosphorylation of the plasma membrane H+-ATPase (Suzuki et al, 1992).…”

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase

“…The PM H+-ATPase contains several potential sites for phosphorylation (Serrano, 1989;Palmgren, 1991), and its phosphorylation has been demonstrated in different conditions (Schaller and Sussman, 1988;Suzuki et al, 1992), but the effects of phosphorylation on the enzyme activity have not yet been unequivocally demonstrated (Zocchi et al, 1985;Suzuki et al, 1992).…”

SYR2, A Gene Necessary for Syringomycin Growth Inhibition of Saccharomyces Cerevisiae

“…The cellular site of action of syringomycin is the plasma membrane. It alters several membrane functions such as membrane potential, protein phosphorylation, H+-ATPase activity and cation-transport fluxes (Zhang & Takemoto, 1987Bidwai e t al., 1987;Suzuki et al, 1992). The relationships of these activities and their relative importance in the response to syringomycin exposure are not clear.…”