Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2.
NGG1p/ADA3p and ADA2p are dual function regulators that stimulate or inhibit a set of yeast transcriptional activator proteins. In vitro, NGG1p and ADA2p associate in a complex that also contains GCN5p (Horiuchi, J., Silverman, N., Marcus, G. A., and Guarente, L. (1995) Mol. Cell. Biol. 15, 1203-1209). We have found that NGG1p and ADA2p are coimmunoprecipitated from yeast whole cell extracts. In fact, <2% of cellular ADA2p was not associated with NGG1p. Also in agreement with their association in vivo, the stability of ADA2p and NGG1p depended on the presence of each other. In addition, three NGG1p- and ADA2p-containing peak fractions were resolved by Q-Sepharose Fast Flow ion-exchange chromatography of whole cell extract. The presence of another high molecular mass complex was supported by the separation of one of the NGG1p- and ADA2p-containing peak fractions by gel-filtration chromatography. Together, the combination of ion-exchange and gel-filtration chromatography suggests a total of four complexes, two with sizes of >2 MDa and single complexes of approximately 900 and 200 kDa. At least one of these complexes was found to associate with the TATA-binding protein (TBP) since TBP was present in immunoprecipitates with NGG1p. The association of TBP with the ADA proteins required amino acids 274-307 of NGG1p, a region of NGG1p required for activity. This supports a role for NGG1p in the interaction with TBP and suggests that the interaction with TBP is functionally relevant.
Saleh
A
A
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Lang
V
V
Cook
R
R
Brandl
C J
CJ
D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005656:Fungal Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*
C090575:ADA2 protein, S cerevisiae; D005656:Fungal Proteins; C083323:NGG1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors