In S. cerevisiae, the accumulation of dysfunctional mitochondria activates a retrograde signal that is mediated through multiple cytosolic regulators. Central to activation is the cytosolic regulator Rtg2p which through its interaction with Mks1p, promotes the nuclear translocation of Rtg1p/3p. Nuclear localized Rtg1p/3p promotes transcription of target genes. Prior work has shown Rtg2p interaction with Mks1p is required for downstream signaling, however the Mks1p binding site within Rtg2p is unknown. To identify this motif, random mutations were generated in RTG2 and a red-white screening strategy was used to assess 14,001 clones. Sequence analysis identified four mutants with amino acid mutations in the carboxy-terminus of Rtg2p that gave rise to defects in CIT2 transcription and loss of Mks1p interaction. Relative to RTG2, all mutants had reduced Rtg2p protein half-lives. Together these results suggest that the carboxy-terminal domain of Rtg2p is essential for retrograde signaling as it may contain the Mks1p binding site.

Rtg2p é uma proteína que participa da sinalização retrógrada, uma via de comunicação da mitocôndria para o núcleo; também tem sido associada com a longevidade em S. cerevisiae. O objetivo deste trabalho foi identificar os determinantes estruturais de Rtg2p, envolvidos na sinalização retrógrada e no envelhecimento. Para isto foram produzidos treze mutantes pontuais a partir do desenho racional por decomposição de redes de correlação de aminoácidos (DRCN). Analisaram-se as cepas mutantes por ensaio de auxotrofia para glutamato, expressão do gene CIT2 e ensaio de longevidade replicativa. Em sua grande maioria as mutações realizadas causaram perturbações nas funções de Rtg2p, com destaque para as cepas E106A, R109E, E137A, T138A e D158A, que apresentaram longevidade igual à da cepa rtg2Δ, com apenas uma mutação pontual. Em conclusão, os resultados obtidos demonstram que o domínio N-terminal é muito importante para a função de Rtg2p, e indicam que existem determinantes estruturais que controlam a longevidade de forma dependente ou independente da resposta retrógrada. / Rtg2p is a protein involved in the retrograde signaling, a pathway of communcation from mitochondria to nucleus; also has been associated with longevity in S. cerevisiae. The goal of this study was to identify the structural determinants of Rtg2p, controlling the function of this protein in retrograde response and aging. For this purpose thirteen point mutants were produced by site-directed mutagenesis, using rational design by decomposition of residues correlation networks (DRCN). The strains was analyzed by glutamate auxotrophy, CIT2 gene expression and replicative life span assays. For the most of performed mutations, generated inactivation to Rtg2p functions, highlighting to R109E, E137A, T138A, and D158A showed longevity equal to rtg2Δ strain, even with a single amino acid change. In conclusion, our results demonstrate that the N-terminal domain is very important to the function of Rtg2p and also show there are structural determinants in Rtg2p that control longevity in both dependent or independent manner of the communication between mitochondria and nucleus.

Rtg2p é uma proteína que participa da sinalização retrógrada, uma via de comunicação da mitocôndria para o núcleo; também tem sido associada com a longevidade em S. cerevisiae. O objetivo deste trabalho foi identificar os determinantes estruturais de Rtg2p, envolvidos na sinalização retrógrada e no envelhecimento. Para isto foram produzidos treze mutantes pontuais a partir do desenho racional por decomposição de redes de correlação de aminoácidos (DRCN). Analisaram-se as cepas mutantes por ensaio de auxotrofia para glutamato, expressão do gene CIT2 e ensaio de longevidade replicativa. Em sua grande maioria as mutações realizadas causaram perturbações nas funções de Rtg2p, com destaque para as cepas E106A, R109E, E137A, T138A e D158A, que apresentaram longevidade igual à da cepa rtg2Δ, com apenas uma mutação pontual. Em conclusão, os resultados obtidos demonstram que o domínio N-terminal é muito importante para a função de Rtg2p, e indicam que existem determinantes estruturais que controlam a longevidade de forma dependente ou independente da resposta retrógrada. / Rtg2p is a protein involved in the retrograde signaling, a pathway of communcation from mitochondria to nucleus; also has been associated with longevity in S. cerevisiae. The goal of this study was to identify the structural determinants of Rtg2p, controlling the function of this protein in retrograde response and aging. For this purpose thirteen point mutants were produced by site-directed mutagenesis, using rational design by decomposition of residues correlation networks (DRCN). The strains was analyzed by glutamate auxotrophy, CIT2 gene expression and replicative life span assays. For the most of performed mutations, generated inactivation to Rtg2p functions, highlighting to R109E, E137A, T138A, and D158A showed longevity equal to rtg2Δ strain, even with a single amino acid change. In conclusion, our results demonstrate that the N-terminal domain is very important to the function of Rtg2p and also show there are structural determinants in Rtg2p that control longevity in both dependent or independent manner of the communication between mitochondria and nucleus.

Unicellular organisms such as yeast have been traditionally studied in shaken cultures, i.e., under condition in which they do not grow attached to solid surfaces as under natural conditions. In nature, cells only rarely live alone, but, on the other hand often create multicellular colonies or biofilms. During last years, yeasts started to be investigated also when grown on solid media. Our laboratory has previously developed special techniques for investigation of yeast colonies. These techniques allowed us to describe individual cell subpopulations within the colonies. The aim of this work was to prepare a series of mutant strains, describe morphology and ultrastructure of their colonies with the aim to contribute to understanding ofthe role of mitochondrial retrograde signalling pathway in the development of yeast colonies. This work describes expression of few selected genes (CIT2, RTG1, RTG2, and RTG3) in colonies of the parental strain BY4742 and of other mutant strains with deletion of one or more genes of RTG regulatory pathways. The results of the diploma thesis together with results of other authors became part of the publication (Podholová et al., 2016). Powered by TCPDF (www.tcpdf.org)