Ambient pH controls glycogen levels by regulating glycogen synthase gene expression in Neurospora crassa. New insights into the pH signaling pathway

PLoS One. 2012;7(8):e44258. doi: 10.1371/journal.pone.0044258. Epub 2012 Aug 31.

Abstract

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacC(KO) strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acids / metabolism
  • Alkalies / metabolism
  • Binding Sites
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression Regulation, Enzymologic*
  • Gene Expression Regulation, Fungal*
  • Gene Knockout Techniques
  • Genes, Fungal / genetics
  • Glycogen / metabolism*
  • Glycogen Synthase / genetics*
  • Glycogen Synthase / metabolism
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Mutation / genetics
  • Neurospora crassa / enzymology*
  • Neurospora crassa / genetics*
  • Neurospora crassa / growth & development
  • Phenotype
  • Promoter Regions, Genetic / genetics
  • Protein Binding / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction / genetics*
  • Stress, Physiological / genetics

Substances

  • Acids
  • Alkalies
  • Fungal Proteins
  • Recombinant Proteins
  • Glycogen
  • Glycogen Synthase

Grant support

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grant nrs. 2008/57566-8 and 2007/07766-8), and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (grant nr. 303973/2009-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.