Abstract
The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256-292 or Δ107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.
Highlights
Methylotrophic yeasts, such as Hansenula polymorpha, Pichia pastoris, and Candida boidinii, are unique yeasts that can utilize methanol as a sole carbon and energy source
Sequence alignment of Hap3p from C. boidinii and S. cerevisiae revealed that, in addition to the conserved N-terminal region, CbHap3p had an extended structure of ca. 190 amino acids long at the C-terminus (Fig. 1)
We showed that strain Cbhap3∆ was impaired in methanol induction during methanol-regulated gene expression (Oda et al, 2015)
Summary
Methylotrophic yeasts, such as Hansenula polymorpha, Pichia pastoris, and Candida boidinii, are unique yeasts that can utilize methanol as a sole carbon and energy source. The promoters of genes encoding methanol-metabolizing enzymes, including alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), glutathione-dependent formaldehyde dehydrogenase (FLD), and formate dehydrogenase (FDH), are highly induced by methanol, and tightly regulated by the presence of alternative carbon sources. These promoters have been used for industrial protein production with methylotrophic yeasts as hosts (Gellissen, 2000; Daly & Hearn, 2005; Yurimoto et al, 2011; Vogl & Glieder, 2012). Methanol-regulated genes are completely repressed in the presence of glucose, which requires. The presence of methanol induces maximum activation of methanol-regulated genes via CbTrm1p (methanol induction; methanol-specific induction) (Hartner & Glieder, 2006; Sasano et al, 2008; Yurimoto, 2009; Sasano et al, 2010; Zhai et al, 2012)
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