Abstract
Rtg1p is a basic helix-loop-helix transcription factor in the yeast Saccharomyces cerevisiae that is required for basal and regulated expression of CIT2, the gene encoding a peroxisomal isoform of citrate synthase. In respiratory incompetent rho degree petite cells, CIT2 transcription is elevated as much as 30-fold compared with respiratory competent rho + cells. Here we provide evidence that Rtg1p interacts directly with a CIT2 upstream activation site (UASr) and that the rho degree/rho + regulation is not due to a change in the levels of Rtg1p. A fusion protein consisting of the DNA binding domain of Gal4p fused to the NH2 terminus of the full-length wild-type Rtg1p was able to transactivate an integrated LacZ reporter under control of the Gal4p-responsive GAL1 UASG in a rho degree/rho(+)-dependent manner. Other Gal4p fusions to deletions or mutations of Rtg1p indicate that the helix-loop-helix domain is essential for transactivation. Regulated expression of CIT2 also requires the RTG2 gene product. The Gal4-Rtg1p fusion was unable to transactivate the LacZ reporter gene in a strain deleted for RTG2, suggesting that the RTG2 product does not act independently of Rtg1p in the rho degree/rho + transcriptional response.
Highlights
§ Supported in part by a fellowship from the American Cancer Society
RTG1 encodes a 177-amino acid protein (Rtg1p) Is Not Limiting in the °/ϩ-responsive Regulation of CIT2 Transcription—The basic helix-loop-helix (bHLH) transcription factor encoded by the RTG1 gene is required for both basal expression of the CIT2 gene as well as for its elevated expression in cells with dysfunctional mitochondria, such as in ° petites [8]
Using Rtg1p-specific antisera to compare protein levels, we found that the amount of Rtg1p did not vary significantly between ϩ and ° cells, nor was its amount affected by a deletion of RTG2, whose product is required for CIT2 expression (Fig. 2)
Summary
§ Supported in part by a fellowship from the American Cancer Society. Present address: Millennium Pharmaceuticals, Inc., 640 Memorial Dr, Cambridge, MA 02139. We have recently found that RTG1 and RTG2 have functions in addition to regulation of CIT2 expression [11]; they are together required for oleic acid-induced expression of genes encoding peroxisomal proteins, as well as for general peroxisome proliferation, which is known to be induced in yeast by oleic acid [12,13,14]. These genes appear to play a central role in a novel three-way organelle communication between mitochondria, the nucleus and peroxisomes. This allows the determination of potential transactivation by Rtg1p independent of its intrinsic DNA binding characteristics
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