Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe

Abstract

An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these 'upstream' git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these 'upstream' git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.

Fig 1

A model for the transcriptional regulation of the S. pombe fbp1 gene. The upstream git genes encode proteins that detect glucose and activate adenylate cyclase, encoded by git2. The git8 gene encodes the alpha subunit of a G protein. Adenylate cyclase produces cAMP, that then activates a cAMP-dependent protein kinase (cAPK), possibly encoded by git6. The kinase acts to inhibit fbp1 transcription. See text for more detail.

Fig 2

cAMP levels in wild type, git2 mutants and a cgs2 mutant strain before and after exposure to glucose. Assays were performed as described in Materials and Methods. (A) Four independent cultures of FWP77 (git⁺) were assayed. The average value at each timepoint was used to produce an average curve (□) that is included in subsequent figures as a reference curve. Standard errors were less than 30% of the value. Also shown are average curves generated from assays of four independent cultures of FWP182 (git2-7; △) and of three independent cultures of FWP184 (git2-61; ◊). (B) Average curve generated from assays of four independent cultures of SP578 (cgs2-2; △). Note that the scales of cAMP concentrations differ between A and B.

Fig 3

cAMP levels in git mutant strains before and after exposure to glucose. Assays were performed as described in Materials and Methods. The FWP77 (git⁺) average curve from Fig. 2A (□) is displayed as a reference curve. (A) Average curves generated from assays of three independent cultures of FWP134 (git1-1; △), of three independent cultures of FWP136 (git3-200; ◊), and of three independent cultures of FWP174 (git5-75; ▲). (B) Average curves generated from assays of four independent cultures of FWP140 (git7-235; △), of three independent cultures of FWP175 (git8-60; ◊), and of three independent cultures of FWP143 (git10-201; ▲). Strain FWP143 was grown in PM containing 0.2% glucose and 3% glycerol as a carbon source (see text).

Fig 4

cAMP levels in a git6 mutant strain before and after exposure to glucose. Assays were performed as described in Materials and Methods. The FWP77 (git⁺) average curve from Fig. 2A (□), is displayed as a reference curve. The average curve generated from assays of three independent cultures of FWP139 (git6-261; ◊) is shown.