Structure/Function Properties of the Yeast Dual Regulator Protein NGG1 That Are Required for Glucose Repression

Abstract

NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p-NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p-independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373).

Identification of Native Complexes Containing the Yeast Coactivator/Repressor Proteins NGG1/ADA3 and ADA2

“…Direct interaction in vitro has been observed between ADA2p and both GCN5p and the carboxyl-terminal 250 amino acids of NGG1p (13,16). Based on the finding that single and double disruptions of ngg1 and ada2 have similar effects on inhibition of GAL4p and PDR1p, we suggested that the same or related ADA complexes are involved in transcriptional activation and repression (9,18). Other coactivator complexes also appear to be involved in the positive and negative regulation of transcription.…”

“…CY946 is a derivative of CY914 that contains a Tn10 LUK disruption of the ADA2 coding region (kindly provided by J. Horiuchi and L. Guarente) (10). CY947 and CY1077 are derivatives of CY946 and CY914, respectively, that contain Myc-tagged NGG1 expressed from the DED1 promoter integrated at his3 (18).…”

“…Western blotting with a primary antibody from ascites fluid derived from the Myc1-9E10 cell line using polyvinylidene difluoride membrane and the SuperSignal chemiluminescence kit (Pierce) has been described (18). Probing with monoclonal anti-HA antibody, TBP polyclonal anti- (26).…”

“…These genes are also required for transactivation by GCN4p (11-13); in fact, GCN5p had been identified because it is required for maximal activation by GCN4p (14). Genetic and in vitro biochemical evidence suggests that the ADA proteins likely act in a complex that contains at least ADA2p, NGG1p, GCN5p,[15][16][17][18]. Direct interaction in vitro has been observed between ADA2p and both GCN5p and the carboxyl-terminal 250 amino acids of NGG1p (13, 16).…”

“…In addition, we have found that TBP coimmunoprecipitates with NGG1p. The significance of the interaction with TBP is suggested by the fact that deletion of amino acids 274 -307 of NGG1p, which results in the slow growth phenotype and loss in repression of GAL4p seen in a ngg1 disruption (18), also results in loss of association with TBP.…”