title: Deglycosylation of cellulosomal enzyme enhances cellulosome assembly in Saccharomyces cerevisiae.
abstract: We have estimated the effects of hyper-mannosylation of dockerin-type cellulase on cellulosome assembly by using Saccharomyces cerevisiae and 44 protein glycosylation mutants, because the heterologous protein displayed on yeast is assumed to be modified by yeast-specific hyper-mannosylation. First, we constructed the yeast strain CtminiCipA, which displays a heterologous scaffolding protein (miniCipA from Clostridium thermocellum) on its cell surface, and glycosylation mutants secreting a dockerin-type cellulase (Cel8Aenz-Cel48Sdoc: a fusion protein of the catalytic domain of C. thermocellum Cel8A and the dockerin domain of C. thermocellum Cel48S). Next, minicellulosomes were assembled by mixing the CtminiCipA strain and the dockerin-type cellulase secreted by each glycosylation mutant. By using an endoglucanase assay and flow cytometric analysis, we showed that some glycosylation mutants enhanced cellulosome assembly; in particular, disruption of glycosylation genes located in the endoplasmic reticulum showed intense enhancement. These findings suggest that inhibition of the core complex or precursor formation in protein glycosylation enhances cellulosome assembly, meaning that absence of glycosylation is more important for cellulosome assembly than reducing the size of the glycochain.
authors.affiliation: Biotechnology Laboratory, Toyota Central R&D Labs Inc., 41-1, Yokomichi, Nagakute, Aichi 480-1192, Japan. e1514@mosk.tytlabs.co.jp
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Takao
authors.initials: T
authors.lastname: Imaeda
authors.forename: Takao
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Katsunori
authors.initials: K
authors.lastname: Kohda
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D044603:Cellulosomes / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002480:Cellulase; D008358:Mannose