Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae.
1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.
Inoue
S B
SB
Department of Mycology, Nippon Roche Research Center, Kamakura, Japan.
Takewaki
N
N
Takasuka
T
T
Mio
T
T
Adachi
M
M
Fujii
Y
Y
Miyamoto
C
C
Arisawa
M
M
Furuichi
Y
Y
Watanabe
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D008565:Membrane Proteins; D010446:Peptide Fragments; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase