| Proper biogenesis and function of mitochondria depend absolutely on the controlled, coordinated synthesis of gene products encoded by both the nuclear and mitochondrial (mt) genomes. Previous studies from this laboratory have shown that under some growth conditions, the rate of mt transcriptional initiation in S. cerevisiae is directly regulated by the cellular cAMP levels, and requires regulated activity of cAMP-dependent protein kinase (PKA). Importantly, an upstream activating sequence (UAS) on the yeast mt genome, that is required for the cAMP-dependent trans-activation event has been identified and characterized. It is the objective of the work described here to identify, clone, and characterize the relevant cAMP-sensitive mt transcriptional trans-activator(s).; The putative yeast mt UAS was used on a one-hybrid screening system to identify two unlinked such ORFs, YMR030W and YJR127C. Expression of each coding sequence is glucose-repressible, and deletion mutants for each gene show a growth defect on glycerol-, but not glucose- or ethanol-, based medium. The specific growth defect in glycerol based medium is explained by the significant attenuated GUT1 and GUT2 transcripts in each single mutant. The transcripts of some mitochondrial related nuclear genes, MRP13 and COX4 genes, are attenuated in each single mutant, but some others are not affected, eg RPO41 and MIP1. Moreover, transcript levels from mt genes that contain the UAS, OLI1 and 21S genes, are attenuated in each single mutant, but this is not the case for a mt gene that does not have a UAS, for example OXI2 gene. A double mutant for the two genes does not grow at all on respiratory carbon sources, and shows a grow problem on glucose-based medium at the point where cells are switching to respiratory growth on the produced ethanol. Mt DNA levels in the single mutants are approximately equal to those of the wild-type parent strain, but the double mutant is devoid of mt DNA. Subcellular localization via GFP-fusion proteins shows that the YMR030W gene product is present in the nucleus and mitochondria, while YJR127C gene product is exclusively present in the nucleus. Thus, the products of the YMR030W and YJR127C coding sequences are required for both respiratory function and maintenance of the mt genome. I designate these two genes RSF1 and RSF2, respectively. |
