Structural and Functional Analysis of Yeast Putative Adaptors

Abstract

Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were cre- ated. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identi- fied regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immuno- precipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for inter- action with ADA3. Deletions of the region that was re- quired for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. More- over, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was dem- onstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interac- tion with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiolog- ically relevant adaptor complex.