Functional complementation analysis of fungal RTG2 homologs in Saccharomyces cerevisiae
Unlu, Ercan Selcuk
AdvisorGordon, M. Donna
Changes in gene expression in response to mitochondrial dysfunction are mediated by components of the retrograde signaling pathway. The mitochondrial signal is recognized and transferred to the nucleus by dynamic interactions between regulatory proteins Rtg2p, Mks1p and Bmh1p. Retrograde signaling genes have been well characterized in the budding yeast Saccharomyces cerevisiae but very little is known about the retrograde response of other fungi. To identify retrograde signaling proteins in other fungi, the protein sequence encoded by the S. cerevisiae RTG2 gene was used to search for fungal homologs using NCBI BlastP and the T-Coffee Multiple Sequence Alignment program. We selected four species having uncharacterized ORFs with more than 66% amino acid identity to Rtg2p for further analysis: Ashbya gossypii, Candida glabrata, Vanderwaltozyma polyspora and Kluyveromyces lactis. In S. cerevisiae, cells deleted for RTG2 are glutamate auxotrophs, and have reduced expression of Aco1p and Cit2p proteins. To determine whether the putative RTG2 genes we identified encode bonefide regulators of the retrograde response pathway, we used standard yeast genetic approaches and molecular biology tools to investigate their ability to complement the defects associated with the rtg2Ä mutant using our S. cerevisiae RTG2 shuffle strain. We investigated functional roles of Rtg2p homologs by comparing Cit2p and Aco1p protein levels, glutamate auxotrophy, as well as analyzing the interaction between Rtg2p homologs and Mks1p. We also analyzed sensitivity of mutant strains under various stress conditions to address possible signaling cross talk between the retrograde signaling pathway and the TOR pathway. Our data show that the fungal Rtg2p homologs from C. glabrata, V. polyspora and K. lactis are functional in mediating the mitochondrial signal through known components of the retrograde signaling cascade. Our immunoprecipitation data suggest that TOR and retrograde signaling may exhibit cross pathway activation under rapamycin treatment. We show that Mks1p, the negative regulator of retrograde signaling pathway is required for Cit2p expression under rapamycin treatment. Given that all Rtg2p homologs showed low affinity for Mks1p which was in turn paralleled by a higher affinity of Mks1p for Bmh1p suggests that Rtg2p may have an additional functional role in influencing the association of Mks1p with Bmh1p.