Purification and characterization of a Ca2+-dependent membrane peptidase involved in the signaling of mating pheromone in Rhodosporidium toruloides.
A mating-type-specific, membrane thiol peptidase (referred to as trigger peptidase) that seems to play a key role in the transmembrane signaling of the lipopeptidyl mating pheromone rhodotorucine A at the cell surface of mating type a cells of Rhodosporidium toruloides (T. Miyakawa, M. Kaji, T. Yasutake, Y.K. Jeong, E. Tsuchiya, and S. Fukui, J. Bacteriol. 162:294-299, 1985) was purified to homogeneity and characterized. The following lines of evidence support the contention that the enzyme we purified was the trigger peptidase: the identical specificity of hydrolysis at the Arg-Asn sequence of rhodotorucine A and the sensitivity of the reaction to sulfhydryl-blocking reagents; the identical specificity for the substrate, with a strict requirement for the presence of the lipid moiety; and the absence of the corresponding activity in the pheromone-producing strain (mating type A) and in a sterile mutant strain, M-39 (type a), that lacks trigger peptidase activity in vivo. The apparent molecular weight of trigger peptidase was estimated to be 68,000 by Sepharose 6B gel filtration in the presence of octylglucoside and 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trigger peptidase alone was inactive but exhibited enzymatic activity with the simultaneous addition of Ca2+, membrane phospholipids, and a nonionic detergent such as octylglucoside. The concentration of Ca2+ required for maximum activation was approximately 1 mM. Only Mn2+ could replace Ca2+ at comparable concentrations. Among the phospholipids tested, only phosphatidylserine and phosphatidylethanolamine supported trigger peptidase activation. Solubilized trigger peptidase was strongly inhibited by antipain and phosphoramidon.
D001487:Basidiomycota / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D000072235:Mating Factor; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism
D009842:Oligopeptides; D010455:Peptides; D000072235:Mating Factor; C017944:rhodotorucine A; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002118:Calcium