Adr1p-dependent regulation of genes encoding FAS II enzymes

The one-year project described herein is concerned with examining the role of the yeast transcription factor Adr1p in regulating genes involved in mitochondrial type II fatty acid synthesis (FAS II). FAS II is proposed to function in human mitochondria, and although a number of human FAS II enzymes have been identified, not much is known about the mechanism controlling their expression. It is important to study the regulation of this mitochondrial process, since dysfunctional mitochondria are a cause of human morbidity and mortality. We propose to study the regulation of the analogous process in yeast mitochondria; Fungal FAS II is critical for mitochondrial function since mutant cells devoid of proteins associated with this biosynthetic pathway are respiratory deficient. FAS II-defective yeast contain only rudimentary mitochondria, fail to assemble cytochrome complexes, and cannot grow on non-fermentable carbon sources. There is convincing evidence that at least one FAS II-enzyme gene, ETR1, is subject to Adr1p regulation. The idea that mitochondrial FAS II might share the same Adr1p-based regulatory circuit as peroxisomal fatty acid ß-oxidation has been invoked previously and is an attractive proposition to initiate a more detailed examination. These envisioned studies should not only shed further light on the regulatory mechanism of fatty acid synthesis in yeast mitochondria, but additionally might lead to the identification of novel circuits that co-regulate the maintenance and expansion of both the mitochondrial and peroxisomal compartments.

Saccharomyces cerevisiae ETR1 encoding 2-trans-enoyl-ACP reductase of mitochondrial type 2 fatty acid synthase (FASII) is subject to Adr1p-dependent transcriptional regulation. Previous genome-wide profiling revealed that ETR1 is 3.2fold more highly expressed in wild-type cells compared to adr1Δ mutant cells. In addition, the ETR1 promoter binds Adr1p in vivo via a resident Adr1p-binding site termed type 1 upstream activation sequence UAS1. Here, Adr1p was examined whether it regulated additional FASII genes. It is important to study FASII regulation since FASII-deficient yeast cells have small underdeveloped mitochondria, fail to assemble cytochrome complexes, and cannot grow on non-fermentable carbon sources or synthesize lipoic acid. The transcriptional regulation of six additional FASII genes was studied using promoter-lacZ fusions monitored in wild-type and adr1 mutant cells grown on rich-glucose medium containing the chromogenic substrate X-gal. The results demonstrated that ACP1, OAR1 and PPT2, whose promoters contain sequences with the potential to represent functional UAS1s, appeared to depend on Adr1p for their expression. In reference to Etr1p, although transcription of the corresponding ETR1 gene is regulated by Adr1p, immunoblotting revealed that in wild-type and adr1Δ mutant cells Etr1p abundance was indistinguishable. Expression in both strains of multiple copies of ETR1 behind the native promoter resulted in similar reductase activities, and the strains` lipoic acid content was equivalent. The combined results cast reasonable doubt on whether transcriptional regulation exerted by Adr1p on ETR1 represents the sole - or most meaningful - layer of control over FASII in wild-type cells. The possibility of additional regulation occurring at the translational is currently being considered, and the untranslated regions of ETR1 and other FASII genes potentially regulated by Adr1p are now being analyzed for involvement in translational control.