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 activ ity of the partially purified enzyme was around 4 mu mol glucose incor porated . min(-1). mg protein(-1). In SDS/PAGE, enrichment of a 200-kD a protein was clearly observed in parallel with the increase in specif ic activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan s ynthase 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 digesti on. 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); hydrop athy profiles of both proteins suggest that these genes encode integra l membrane proteins which can be assumed to have approximately 16 tran smembrane domains. Disruption of each gene was not lethal, but disrupt ion of both genes was lethal, The 1,3-beta-D-glucan synthase activitie s of membrane and partially purified enzyme of gsc1::URA3 cells were s ignificantly lower than those of the wild-type and gsc2::LEU2 cells.