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

Abstract

The Hap complex of the methylotrophic yeast 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 . Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from (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 Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Δ 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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000275
2016-05-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/5/898.html?itemId=/content/journal/micro/10.1099/mic.0.000275&mimeType=html&fmt=ahah

References

  1. Baxevanis A. D., Arents G., Moudrianakis E. N., Landsman D. 1995; A variety of DNA-binding and multimeric proteins contain the histone fold motif. Nucleic Acids Res 23:2685–2691 [View Article][PubMed]
    [Google Scholar]
  2. Bourgarel D., Nguyen C. C., Bolotin-Fukuhara M. 1999; HAP4, the glucose-repressed regulated subunit of the HAP transcriptional complex involved in the fermentation-respiration shift, has a functional homologue in the respiratory yeast Kluyveromyces lactis . Mol Microbiol 31:1205–1215 [View Article][PubMed]
    [Google Scholar]
  3. Brachmann C. B., Davies A., Cost G. J., Caputo E., Li J., Hieter P., Boeke J. D. 1998; Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132 [View Article][PubMed]
    [Google Scholar]
  4. Buschlen S., Amillet J. M., Guiard B., Fournier A., Marcireau C., Bolotin-Fukuhara M. 2003; The S. cerevisiae HAP complex, a key regulator of mitochondrial function, coordinates nuclear and mitochondrial gene expression. Comp Funct Genomics 4:37–46 [View Article][PubMed]
    [Google Scholar]
  5. Chodosh L. A., Olesen J., Hahn S., Baldwin A. S., Guarente L., Sharp P. A. 1988; A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable. Cell 53:25–35 [View Article][PubMed]
    [Google Scholar]
  6. Daly R., Hearn M. T. 2005; Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138 [View Article][PubMed]
    [Google Scholar]
  7. Forsburg S. L., Guarente L. 1989; Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev 3:1166–1178 [View Article][PubMed]
    [Google Scholar]
  8. Gellissen G. 2000; Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750 [View Article][PubMed]
    [Google Scholar]
  9. Hartner F. S., Glieder A. 2006; Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact 5:39 [View Article][PubMed]
    [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. 1983; Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168[PubMed]
    [Google Scholar]
  11. McNabb D. S., Pinto I. 2005; Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA complex in Saccharomyces cerevisiae . Eukaryot Cell 4:1829–1839 [View Article][PubMed]
    [Google Scholar]
  12. McNabb D. S., Tseng K. A., Guarente L. 1997; The Saccharomyces cerevisiae Hap5p homolog from fission yeast reveals two conserved domains that are essential for assembly of heterotetrameric CCAAT-binding factor. Mol Cell Biol 17:7008–7018 [View Article][PubMed]
    [Google Scholar]
  13. Oda S., Yurimoto H., Nitta N., Sasano Y., Sakai Y. 2015; Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii . Eukaryot Cell 14:278–285 [View Article][PubMed]
    [Google Scholar]
  14. Ramil E., Agrimonti C., Shechter E., Gervais M., Guiard B. 2000; Regulation of the CYB2 gene expression: transcriptional co-ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors. Mol Microbiol 37:1116–1132 [View Article][PubMed]
    [Google Scholar]
  15. Ridenour J. B., Bluhm B. H. 2014; The HAP complex in Fusarium verticillioides is a key regulator of growth, morphogenesis, secondary metabolism, and pathogenesis. Fungal Genet Biol 69:52–64 [View Article][PubMed]
    [Google Scholar]
  16. Romier C., Cocchiarella F., Mantovani R., Moras D. 2003; The NF-YB/NF-YC structure gives insight into DNA binding and transcription regulation by CCAAT factor NF-Y. J Biol Chem 278:1336–1345 [View Article][PubMed]
    [Google Scholar]
  17. Sakai Y., Kazarimoto T., Tani Y. 1991; Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5′-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation. J Bacteriol 173:7458–7463[PubMed]
    [Google Scholar]
  18. Sakai Y., Akiyama M., Kondoh H., Shibano Y., Kato N. 1996; High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system. Biochim Biophys Acta 1308:81–87 [CrossRef]
    [Google Scholar]
  19. Sasano Y., Yurimoto H., Yanaka M., Sakai Y. 2008; Trm1p, a Zn(II)2Cys6-type transcription factor, is a master regulator of methanol-specific gene activation in the methylotrophic yeast Candida boidinii . Eukaryot Cell 7:527–536 [View Article][PubMed]
    [Google Scholar]
  20. Sasano Y., Yurimoto H., Kuriyama M., Sakai Y. 2010; Trm2p-dependent derepression is essential for methanol-specific gene activation in the methylotrophic yeast Candida boidinii . FEMS Yeast Res 10:535–544[PubMed]
    [Google Scholar]
  21. Singh R. P., Prasad H. K., Sinha I., Agarwal N., Natarajan K. 2011; Cap2-HAP complex is a critical transcriptional regulator that has dual but contrasting roles in regulation of iron homeostasis in Candida albicans . J Biol Chem 286:25154–25170 [View Article][PubMed]
    [Google Scholar]
  22. Sybirna K., Guiard B., Li Y. F., Bao W. G., Bolotin-Fukuhara M., Delahodde A. 2005; A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae . Curr Genet 47:172–181 [View Article][PubMed]
    [Google Scholar]
  23. Sybirna K., Petryk N., Zhou Y. F., Sibirny A., Bolotin-Fukuhara M. 2010; A novel Hansenula polymorpha transcriptional factor HpHAP4-B, able to functionally replace the S. cerevisiae HAP4 gene, contains an additional bZip motif. Yeast 27:941–954 [View Article][PubMed]
    [Google Scholar]
  24. Tani Y., Sakai Y., Yamada H. 1985; Isolation and characterization of a mutant of a methanol yeast, Candida boidinii S2, with higher formaldehyde productivity. Agric Biol Chem 49:2699–2706 [View Article]
    [Google Scholar]
  25. Tanoue S., Kamei K., Goda H., Tanaka A., Kobayashi T., Tsukagoshi N., Kato M. 2006; The region in a subunit of the Aspergillus CCAAT-binding protein similar to the HAP4p-recruiting domain of Saccharomyces cerevisiae Hap5p is not essential for transcriptional enhancement. Biosci Biotechnol Biochem 70:782–787 [View Article][PubMed]
    [Google Scholar]
  26. Vogl T., Glieder A. 2013; Regulation of Pichia pastoris promoters and its consequences for protein production. N Biotechnol 30:385–404 [View Article][PubMed]
    [Google Scholar]
  27. Wach A. 1996; PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae . Yeast 12:259–265 [View Article][PubMed]
    [Google Scholar]
  28. Xing Y., Fikes J. D., Guarente L. 1993; Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain. EMBO J 12:4647–4655[PubMed]
    [Google Scholar]
  29. Yurimoto H. 2009; Molecular basis of methanol-inducible gene expression and its application in the methylotrophic yeast Candida boidinii . Biosci Biotechnol Biochem 73:793–800 [View Article][PubMed]
    [Google Scholar]
  30. Yurimoto H., Oku M., Sakai Y. 2011; Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis. Int J Microbiol 2011:101298 [View Article][PubMed]
    [Google Scholar]
  31. Zhai Z., Yurimoto H., Sakai Y. 2012; Molecular characterization of Candida boidinii MIG1 and its role in the regulation of methanol-inducible gene expression. Yeast 29:293–301 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000275
Loading
/content/journal/micro/10.1099/mic.0.000275
Loading

Data & Media loading...

Supplements

Supplementary Data

PDF

Most cited Most Cited RSS feed