Rtg3p, a basic helix-loop-helix/leucine zipper protein that functions in mitochondrial-induced changes in gene expression, contains independent activation domains

Rtg3p and Rtg1p are basic helix-loop-helix/leucine zipper protein transcription factors in yeast that interact and bind to sites in an upstream activation sequence element in the 5'-flanking region of CIT2, a gene encoding a peroxisomal isoform of citrate synthase. These factors are required both for basal expression of CIT2 and its elevated expression in cells with dysfunctional mitochondria, such as in respiratory-deficient petite cells lacking mitochondrial DNA (ρ°). This elevated expression of CIT2 is called the retrograde response. Here we show that fusion constructs between the Gal4p DNA binding domain and Rtg3p transactivate the expression of a LacZ reporter gene under the control of a GAL1 promoter element. We have identified two activation domains in Rtg3p: a strong carboxyl-terminal domain from amino acids 375-486, and a weaker amino-terminal domain from amino acids 1-175: neither of these activation domains contain the bHLH/Zip motif. We have also identified a serine/threonine-rich domain of Rtg3p within amino acids 176-282 that is inhibitory to transactivation. In addition, the transcriptional activity of the Gal4-Rtg3p fusion proteins does not require either Rtg1p or Rtg2p; the latter is a protein containing an hsp70-like ATP binding domain that is also necessary for CIT2 expression. In contrast, transcriptional activation by Gal4-Rtg1p fusion proteins requires the Rtg1p basic helix-loop-helix/leucine zipper protein domain, as well as Rtg3p and Rtg2p. These data suggest that transcriptional activation by the Rtg1p-Rtg3p complex is largely the function of Rtg3p. Experiments are also presented suggesting that Rtg3p is limiting for gene expression in respiratory- competent (ρ⁺) cells.