Characterization and Gene Cloning of 1,3-beta-D-Glucan Synthase from Saccharomyces Cerevisiae

Abstract

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.

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

“…Several (1,3)-b-D-glucan synthases from fungi contain components of comparable size to the 190-kDa polypeptide associated with pollen-tube CalS, with reports of polypeptides of 165 kDa in Neurospora (Awald et al 1993), approximately 200 kDa in Aspergillus (Kelly et al 1996), and 200 kDa in Saccharomyces (Inoue et al 1995). These cases therefore give precedent for large, intramembrane polypeptides being associated with or responsible for glucan synthesis.…”

“…This process is a function of the synthase catalytic activity, speci®cally targeting the enzyme complex and having the potential to provide substantial levels of puri®cation: 150-fold and 700-fold enrichment have been reported from peas and Saccharomyces respectively (Dhugga and Ray 1994;Inoue et al 1995). In the present study, entrapment of the Ca 2+ -independent pollen-tube CalS could be carried out in the presence of EDTA, which reduced non-speci®c precipitation, but a lower degree of puri®cation (generally 10-fold) was achieved.…”

Mutations that are synthetically lethal with a gas1Δ allele cause defects in the cell wall of Saccharomyces cerevisiae

“…The b-1,3-glucan synthase is a plasma membrane complex made up of a catalytic subunit (Fks1p or Fks2p) (Douglas et al 1994;Inoue et al 1995) and the regulatory subunit (Rho1p) (Drgonova et al 1996;Qadota et al 1996). The b-1,3-glucan becomes alkali-insoluble and acquires further structural strength by forming covalent linkages with chitin; a b-1,4-linked polymer of N -acetylglucosamine (Kollar et al 1995.…”

Biosynthesis of Plant Cell Wall and Related Polysaccharides by Enzymes of the GT2 and GT48 Families

“…The identification of the gene family that encodes the callose synthases came via an indirect route, from work on the genes responsible for the synthesis of a (1,3) -β -d -glucan in fungi. In the yeast Saccharomyces cerevisiae , the Fks ( FK 506 s upersensitive) genes are believed to be involved in (1,3) -β -d -glucan biosynthesis (Douglas et al 1994 ;Cabib et al 2001 ;Dijkgraaf et al 2002 ), as shown by mutant analyses (Douglas et al 1994 ;Inoue et al 1995 ) and biochemical studies (Inoue et al 1995 ). Searches of EST databases for plant genes with similarity to the Fks genes led to the identifi cation of a family of candidate genes, called glucan synthase -like ( GSL ) genes (Saxena & Brown 2000 ;Cui et al 2001b ).…”