Abstract
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 (rho degrees ). 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 (rho+) cells.
Highlights
Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through changes in nuclear gene expression [1,2,3,4,5]
We have found that CIT2 transcription is activated as much as 30-fold in cells that have blocks in the tricarboxylic acid (TCA) cycle or are respiratorydeficient, for example, because of a complete loss of mitochondrial DNA [7, 11, 12]
Rtg1p has some unusual features for a bHLH protein; in addition to an unusually long loop domain of some 39 amino acid residues, it has a very short basic DNA binding domain that lacks certain conserved amino acids found in the basic region of most other bHLH proteins
Summary
Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through changes in nuclear gene expression [1,2,3,4,5]. Rtg1p has some unusual features for a bHLH protein; in addition to an unusually long loop domain of some 39 amino acid residues, it has a very short basic DNA binding domain that lacks certain conserved amino acids found in the basic region of most other bHLH proteins. Those residues are known to make specific base contacts in the major groove of target site DNA (19 –21). In wild-type cells, the Gal4-Rtg1p chimera was able to activate transcription of an integrated LacZ reporter gene under the control of a GAL1 promoter element (UASG). We suggest that Rtg3p may be limiting for CIT2 expression in respiratory-competent (r1) cells and that Rtg2p could function to recruit Rtg3p for interaction with Rtg1p
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