The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin.
Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Delta : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-beta-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-beta-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-beta-glucan synthase.
Miyamoto
Masahiko
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashizima, Niigata 956-8603, Japan.
Onozato
Naohiko
N
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashizima, Niigata 956-8603, Japan.
Selvakumar
Dakshnamurthy
D
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashizima, Niigata 956-8603, Japan.
Kimura
Tetsuya
T
Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
Furuichi
Yasuhiro
Y
GeneCare Research Institute Co. Ltd, Kamakura 247-0063, Japan.
Komiyama
Tadazumi
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashizima, Niigata 956-8603, Japan.
D002176:Candida albicans / Q000254:growth & development; D002462:Cell Membrane / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D055646:Killer Factors, Yeast; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009183:Mycotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000254:growth & development
D055646:Killer Factors, Yeast; D009183:Mycotoxins; D006639:Histidine; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D004047:Diethyl Pyrocarbonate