Mylin L M, Johnston M, Hopper J E
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Mol Cell Biol. 1990 Sep;10(9):4623-9. doi: 10.1128/mcb.10.9.4623-4629.1990.
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.
GAL4I、GAL4II和GAL4III是酵母转录激活蛋白的三种形式，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳过程中，很容易根据电泳迁移率将它们区分开来。磷酸化导致迁移最慢的形式GAL4III迁移率降低，发现它与高水平的GAL/MEL基因表达密切相关（L. 米林、P. 巴特和J. 霍珀，《基因与发育》3:1157 - 1165，1989年）。在此我们表明，与GAL4III一样，GAL4II经磷酸酶处理后可转化为GAL4I，这表明在体内GAL4II是由GAL4I磷酸化而来。我们发现，在GAL4驱动GAL/MEL基因表达水平适中至较低的条件下过量产生GAL4的细胞，只显示出GAL4I和GAL4II形式。为了区分在没有GAL4III的情况下，哪种形式的GAL4（GAL4I、GAL4II或两者）可能负责转录激活，我们对从四个因无法激活转录而选择的gal4错义突变体中具有UASgal结合能力的GAL4蛋白进行了免疫印迹分析（M. 约翰斯顿和J. 多弗，《美国国家科学院院刊》84:2401 - 2405，1987年；《遗传学》120:63 - 74，1988年）。三个未检测到GAL1表达的突变体似乎没有形成GAL4II或GAL4III，但恢复了GAL4依赖性转录的回复突变体确实显示出类似GAL4II或GAL4III的电泳条带。在一个UASgal结合突变体中检测到GAL4II，这表明形成GAL4II既不需要UASgal结合，也不需要GAL/MEL基因激活。总体而言，我们的结果表明GAL4I在转录激活中可能是无活性的，而GAL4II似乎是有活性的。鉴于这项工作，我们推测GAL4I的磷酸化使其能够激活转录。
