Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer.
ABSTRACT The CYC1 gene of Saccharomyces cerevisiae is positively regulated by the HAP2 and HAP3 proteins, which form a heteromeric complex that binds to a CCAAT box in the upstream activation site, UAS2, and which activate transcription in a nonfermentable carbon source. We carried out a genetic analysis to identify additional trans-acting regulatory factors exerting their effects through UAS2. We present the identification and characterization of a new locus, HAP4, which is shown to encode a subunit of the DNA-binding complex at UAS2. In the hap4 mutant, the binding of HAP2 and HAP3 (HAP2/3) is not observed in vitro. The HAP4 gene is regulated transcriptionally by a carbon source, suggesting that it encodes a regulatory subunit of the bound complex. The sequence of HAP4 shows a highly acidic region, which innactivated the protein when deleted. Replacement of this region with the activation domain of GAL4 restored activity, suggesting that it provides the principal activation domain to the bound HAP2/3/4 complex.
Full-textDOI: · Available from: Susan Forsburg, Jun 05, 2015
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ABSTRACT: Dietary restriction (DR) attenuates many detrimental effects of aging and consequently promotes health and increases longevity across organisms. While over the last 15 years extensive research has been devoted towards understanding the biology of aging, the precise mechanistic aspects of DR are yet to be settled. Abundant experimental evidence indicates that the DR effect on stimulating health impinges several metabolic and stress-resistance pathways. Downstream effects of these pathways include a reduction in cellular damage induced by oxidative stress, enhanced efficiency of mitochondrial functions and maintenance of mitochondrial dynamics and quality control, thereby attenuating age-related declines in mitochondrial function. However, the literature also accumulates conflicting evidence regarding how DR ameliorates mitochondrial performance and whether that is enough to slow age-dependent cellular and organismal deterioration. Here, we will summarize the current knowledge about how and to which extent the influence of different DR regimes on mitochondrial biogenesis and function contribute to postpone the detrimental effects of aging on healthspan and lifespan. Copyright © 2015. Published by Elsevier B.V.Biochimica et Biophysica Acta 05/2015; DOI:10.1016/j.bbabio.2015.05.005 · 4.66 Impact Factor
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ABSTRACT: Flavohemoglobins are the main detoxifiers of nitric oxide (NO) in bacteria and fungi and are induced in response to nitrosative stress. In fungi, the flavohemoglobin encoding gene YHB1 is positively regulated by transcription factors which are activated upon NO exposure. In this study, we show that in the model yeast Saccharomyces cerevisiae and in the human pathogen Candida glabrata, the transcription factor Yap7 constitutively represses YHB1 by binding its promoter. Consequently, YAP7 deletion conferred high NO resistance to the cells. Co-immunoprecipitation experiments and mutant analyses indicated that Yap7 represses YHB1 by recruiting the transcriptional repressor Tup1. In S. cerevisiae, YHB1 repression also involves interaction of Yap7 with the Hap2/3/5 complex through a conserved Hap4-like-bZIP domain, but this interaction has been lost in C. glabrata. The evolutionary origin of this regulation was investigated by functional analyses of Yap7 and of its paralogue Yap5 in different yeast species. These analyses indicated that the negative regulation of YHB1 by Yap7 arose by neofunctionalization after the whole genome duplication which led to the C. glabrata and S. cerevisiae extant species. This work describes a new aspect of the regulation of fungal nitric oxidase and provides detailed insights into its functioning and evolution. This article is protected by copyright. All rights reserved.Molecular Microbiology 03/2015; 96(5). DOI:10.1111/mmi.12983 · 5.03 Impact Factor
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ABSTRACT: We 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 gene disrupted strains, Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ, showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, transcriptional activities of methanol-inducible promoters significantly decreased compared to the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analysis 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell 01/2015; 14(3). DOI:10.1128/EC.00285-14 · 3.18 Impact Factor