Phosphorylated forms of GAL4 are correlated with ability to activate transcription.
GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.
Mylin
L M
LM
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Johnston
M
M
Hopper
J E
JE
D000483:Alleles; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D014644:Genetic Variation; D005838:Genotype; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors