title: Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro.
abstract: The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to beta(1-6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.
authors.affiliation: Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA. enricoc@bdg10.niddk.nih.gov
authors.forename: Enrico
authors.initials: E
authors.lastname: Cabib
authors.forename: Vladimir
authors.initials: V
authors.lastname: Farkas
authors.forename: Ondrej
authors.initials: O
authors.lastname: Kosík
authors.forename: Noelia
authors.initials: N
authors.lastname: Blanco
authors.forename: Javier
authors.initials: J
authors.lastname: Arroyo
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
mesh_terms: D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry; D004072:Digitonin / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D008957:Models, Genetic; D009844:Oligosaccharides / Q000737:chemistry; D010539:Permeability; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D005656:Fungal Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D002686:Chitin; C407735:CRH1 protein, S cerevisiae; D006026:Glycoside Hydrolases; C407734:UTR2 protein, S cerevisiae; D002688:Chitinases; D004072:Digitonin