Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor

Abstract

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROXl factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using ZacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROXl factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROXl factor.The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADPIATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX%, HEM13 and CYC7 that are negatively regulated by oxygen and heme.In Saccharomyces cerevisiae the mitochondria1 ADP/ATP translocator exists as three isozymes that are encoded by distinct nuclear genes, AACI, AAC2, and AAC3 [l-31. The reason for the existence of multiple genes encoding a protein functioning in ADP/ATP translocation across mitochondrial membrane is not apparent. Gene-disruption experiments have shown that the product of AAC2 gene is the major protein involved in ADP/ATP exchange across the mitochondrial membrane and that it is essential for oxidative phosphorylation [2-41. The disruption of the AACl or AAC3 gene did not yield a distinct phenotype, but the combination of these mutations with other nuclear mutations resulted in phenotypes indicating a vacuolar role for the AACZ product and a role in the anaerobic metabolism for the AAC3 product [3, It has been shown that AAC genes are differentially regulated to express separate isofoms under different physiologi-51. and that a functional ADP/ATP translocator is essential for anaerobic growth of yeast cells [9].The expression of several other yeast genes, TIFSIB (ANBI) [lo, 161, COX% [ll, 121, CYC7 [131, HEM13 [141 and ERG11 [15] is negatively regulated by oxygen. These genes encode proteins functioning in various metabolic pathways including translation initiation (TIFSZB), mitochondrial respiratory chain (CYC7, COX%), heme (HEMZ3) and ergosterol (ERGll) biosynthesis. The oxygen repression in most of these genes appears to be mediated through the action of ROXl factor, the expression of which is heme dependent [ 17, 181. Deletion analysis and site-directed mutagenesis of TIF.51 B promoter pinpointed the upstream sequences which are most probably involved in the negative control of TIFSIB gene [19, 201. Similar sequences are also present in the upstream regions of the o...

Expression of the AAC2 Gene Encoding the Major Mitochondrial ADP/ATP Carrier in Saccharomyces cerevisiae is Controlled at the Transcriptional Level by Oxygen, Heme and HAP2 Factor

“…1 B). Similarly, the mutation in the ROXl gene, which derepressed the AAC3 gene [9], did not influence the cellular content of AAC2 protein (not shown).…”

“…A SnaBISspI restriction fragment (-380 to -179 bp) containing the AAC2 UAS was cloned into the EcoRI-blunted site of the centromeric plasmid pRSA3s in front of the -93/+269 bp AAC3 fragment fused to the lac2 gene [9]. The orientation of the ligated SnaBI-SspI fragment was determined by digestion of the recombinant plasmid with ApaI restriction endonuclease, which cleaves at -219 bp within AAC2 promoter region.…”

“…To confirm that this region is functioning as an UAS we tested the ability of AAC2 upstream DNA to activate transcription of another AAC 'iso-gene' lacking its own regulatory sequences. For this purpose AAC2 DNA between -380 and -180 (SnaBI-SspI fragment) was placed in front of the 'hypoxic' AAC3 gene containing only 93 bp upstream from its translation initiation codon and fused to lac2 [9]. To enable the cloning of the UAS, the inserted DNA fragment contains at the 3'-end additional sequences up to the SspI restriction side.…”

“…While the AACl and AAC2 genes are expressed preferentially under derepressed conditions in the presence of oxygen, the AAC3 gene is expressed only under anaerobic conditions [7]. Previous work [9] demonstrated that the oxygen repression of AAC3 gene is mediated by heme, ROXl transcription factor, and by an upstream repression site (URS) present at the 5'-end of the gene. Both the AACl and AAC3 genes encode enzymically functional proteins but they are very weakly expressed [7,101.…”

Adenine nucleotide translocase family: four isoforms for apoptosis modulation in cancer

“…The expression of human ANT2 gene in highly proliferative cells is governed by glycolysis-regulated box, a transcriptional element involved in the regulation of glycolytic ATP import into mitochondria (Giraud et al, 1998). Under aerobic conditions, the specific ROX1 factor binds to an ROX1-binding motif in the ANT2 promoter region and the ANT2 gene is regulated through heme that blocks its expression (Sabova´et al, 1993). Sequence comparison of the human ANT and the three yeast AAC promoters led to the identification of a 13 bp motif in the ANT2 promoter: CATTGTTATGATT (nt À1196 to À1184 relative to the transcription start) homologous to an AAC3 promoter sequence.…”