title: Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha.
abstract: The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.
authors.affiliation: Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv.
authors.forename: V M
authors.initials: VM
authors.lastname: Ubiyvovk
authors.forename: O V
authors.initials: OV
authors.lastname: Blazhenko
authors.forename: M
authors.initials: M
authors.lastname: Zimmermann
authors.forename: M J
authors.initials: MJ
authors.lastname: Sohn
authors.forename: H A
authors.initials: HA
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D002104:Cadmium / Q000378:metabolism / Q000633:toxicity; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D005816:Genetic Complementation Test; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism / Q000633:toxicity; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013431:Sulfates / Q000378:metabolism / Q000494:pharmacology; D013463:Sulfuric Acid Esters / Q000378:metabolism / Q000494:pharmacology
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D013431:Sulfates; D013463:Sulfuric Acid Esters; D002104:Cadmium; D008769:Methylnitronitrosoguanidine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase; C490862:GSH1 protein, S cerevisiae; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine; D000432:Methanol