title: The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction.
abstract: Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.
authors.affiliation: Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.
authors.forename: Naomi
authors.initials: N
authors.lastname: Ono
authors.affiliation: Division of Life Science, Graduate School of Agricultural Science, Tohoku University,Aoba-ku, Sendai 981-8555, Japan2.|Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.
authors.forename: Tomio
authors.initials: T
authors.lastname: Yabe
authors.affiliation: Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sudoh
authors.affiliation: Division of Life Science, Graduate School of Agricultural Science, Tohoku University,Aoba-ku, Sendai 981-8555, Japan2.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Nakajima
authors.affiliation: Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan3.
authors.forename: Toshiko
authors.initials: T
authors.lastname: Yamada-Okabe
authors.affiliation: Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.
authors.forename: Mikio
authors.initials: M
authors.lastname: Arisawa
authors.affiliation: Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.
authors.forename: Hisafumi
authors.initials: H
authors.lastname: Yamada-Okabe
mesh_terms: D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002687:Chitin Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D002793:Cholic Acids / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D002352:Carrier Proteins; D002788:Cholesterol Esters; D002793:Cholic Acids; D003902:Detergents; D005656:Fungal Proteins; C083185:SKT5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002687:Chitin Synthase; D014357:Trypsin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C013440:cholesteryl succinate