Abstract
The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.
Highlights
The budding yeast Saccharomyces cerevisiae prefers to ferment glucose even when oxygen is available [1,2,3]
We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function
We have presented three pieces of evidence that support the view that PKA contributes to glucose induction of HXT gene expression by catalyzing phosphorylation of Rgt1
Summary
The rgt1⌬ cells (FM557) were cotransformed with the PCR products (which overlap by the length of the PCR primers) and a URA3-containing plasmid that carries lexA (pSH2-1) [28], linearized by digestion with BamHI, selecting for Uraϩ cells. The PCR products are incorporated into the plasmid by homologous recombination regenerating Rgt (but with the mutation changing Ser to Ala). Cell lysates were sonicated five times with 10-s pulses using a microtip. Proteins with their attached genomic DNA fragments, which averaged 200 –500 bp in length, were precipitated with mouse monoclonal anti-LexA antibodies (Santa Cruz Biotechnology). The sequences of the PCR primer pair used to detect the HXT1 promoter were 5Ј-ATATA-
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