Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

Abstract

cWe identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2⌬, Cbhap3⌬, and Cbhap5⌬ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

“…The ScHap2p/3p/4p/5p complex activates genes involved in respiratory metabolism and mitochondria biogenesis, and is indispensable for respiratory growth on non-fermentable carbon sources. Although CbHap2p/3p/5p proteins were found to be necessary for maximum activation of methanol-regulated genes and growth on methanol in C. boidinii, the CbHap complex was not necessary for growth on non-fermentable carbon sources or for derepression (Oda et al, 2015).…”

“…The interaction of CbHap3p and CbHap5p was investigated as described previously (Oda et al, 2015). C. boidinii strains expressing internal amino-acids-deleted CbHap3p-HA (haemagglutinin) were constructed as follows.…”

“…C. boidinii strains expressing internal amino-acids-deleted CbHap3p-HA (haemagglutinin) were constructed as follows. PCR was performed with primers CbHAP3-100E-up/CbHAP3-225E-down, CbHAP3-97K-up/CbHAP3-225E-down and CbHAP3-85E-up/CbHAP3-225E-down using pP ACT1 -CbHAP3-HA (Oda et al, 2015) as a template, yielding pCbHAP3-HAD101-224, pCbHAP3-HAD98-224 and pCbHAP3-HAD86-224, respectively. The resulting plasmids were linearized with EcoT22I and used to transform the Cbhap3Dura3 strain.…”

“…Then, the 7.8 kb Pst I fragment of pP ACT1 -ScHAP3-end and the 1.7 kb Pst I fragment of the coding region of GFP were ligated to yield pP ACT1 -ScHAP3-GFP. pP ACT1 -ScHAP3, pP ACT1 -ScHAP3-GFP and pP ACT1 -ScCbHAP3 were linearized with EcoT22I, and used to transform strain Cbhap3Dura3 (Oda et al, 2015). The resulting strains were named ScHAP3/Cbhap3D, ScHAP3-GFP/Cbhap3D and Sc-CbHAP3/Cbhap3D, respectively.…”

“…In a previous study, we identified a multimeric transcription factor, the CbHap complex, which is involved in methanolregulated gene expression, specifically methanol induction (Oda et al, 2015). The Hap complex is highly conserved amongst all eukaryotes, from yeasts to humans (Ramil et al, 2000;McNabb & Pinto, 2005;Sybirna et al, 2005;Singh et al, 2011;Ridenour & Bluhm, 2014).…”