Title

Phosphorylated Forms of GAL4 Are Correlated Wtih Ability to Activate Transcription

Document Type

Article

Publication Date

1990

Abstract

GAL4(I), GAL4(II), and GAL4(III) 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, GAL4(III), 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 GAL4(II), like GAL4(III), can be converted to GAL4(I) by phosphatase treatment, suggesting that in vivo GAL4(II) is derived from GAL4(I) 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 GAL4(I) and GAL4(II). To distinguish which forms of GAL4 (GAL4(I), GAL4(II), or both) might be responsible for transcription activation in the absence of GAL4(III), we performed immunoblot analysis of 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 GAL4(II) or GAL4(III), but revertants in which GAL4-dependent transcription was restored did display GAL4(II)- or GAL4(III)-like electrophoretic species. Detection of GAL4(II) in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4(II). Overall, our results imply that GAL4(I) may be inactive in transcriptional activation, whereas GAL4(II) appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4(I) makes it competent to activate transcription.

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