Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator.
In yeast, galactose triggers a rapid GAL4-dependent induction of galactose/melibiose regulon (GAL/MEL) gene transcription, and glucose represses this activation. We discovered that alterations in the physical state of the GAL4 protein correlate with activation and repression of the GAL/MEL genes. Using Western immunoblot assay, we observe two electrophoretic forms of GAL4 protein-GAL4I and GAL4II-in noninduced cells. In the absence of glucose, the addition of galactose to such cells results in the rapid appearance of a third and slower-migrating form, GAL4III, which differs from at least GAL4I by phosphorylation. GAL80-deletion cells that constitutively transcribe galactose-responsive genes due to the lack of the GAL80 protein, an antagonist of the GAL4 protein, exhibit GAL4III without galactose addition. Addition of glucose, which results in rapid repression of galactose gene transcription, triggers a rapid elimination of GAL4III and an increase in GAL4II. Cycloheximide experiments provide evidence that the galactose- and glucose-triggered GAL4 protein mobility shifts are due to post-translational modification. GAL4III is labeled with [32P]phosphate in vivo; in vivo 35S-labeled GAL4III could be converted by phosphatase treatment in vitro to GAL4I. We present a model proposing that phosphorylation state changes in the GAL4 protein are key to modulating its activity.
Mylin
L M
LM
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Bhat
J P
JP
Hopper
J E
JE
D000469:Alkaline Phosphatase / Q000494:pharmacology; D002872:Chromosome Deletion; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000469:Alkaline Phosphatase; D005947:Glucose; D005690:Galactose