Influência do metabolismo de carbono e do nível de oxigênio sobre a localização da permease de lactose em Kluyveromyces lactis

In this work we analyzed the influence of the carbon source, oxygen level and snf1 mutation on the subcelular localization of Lac12GFP permease in the yeast Kluyveromyces lactis. Initially we showed that K. lactis has higher affinity for lactose than for glucose. Our data suggest that the KlSnf1p dependent subcelular localization of Lac12GFP permease was not affected by glucose. Furthermore, we observed that the subcelular localization of this permease was similar when K. lactis was grown on galactose and glucose, but different on lactose. The mRNA levels of the LAC12GFP gene expressed from KlSnf1p independent promoter were similar between the wild type and Klsnf1 mutant, indicating that KlSnf1p markedly affects the LAC12GFP gene expression only at post- translational level. Analysis of the subcelular localization of lactose permease in Klsnf1 mutant showed that the permease is found intracellularly at the vacuole membrane, suggesting that Klsnf1p may be involved with the lactose permase translocation into vacuole. In hypoxic condition, more lactose permease was localized at plasma membrane up to 3 hours of growth, indicating that the oxygen availability influences the localization of Lac12GFPp. Furthermore, we performed a screening (multicopy suppression) to identify negative regulators of lactose uptake in K. lactis. The multicopy suppression was used to select transformants able to suppress lactose toxicity, because lactose is toxic for Kllac4 mutant grown on minimal medium containing lactose plus glycerol as a carbon source. Among the 68 plasmids rescued from WBS1 (lac4 mutant) transformed with a Kep6-based genomic plasmid library (multicopy suppression), nine were bigger than Kep6 (empty vector). Nevertheless, our data showed that the suppression was due to a spontaneous mutation. In these mutants the lactose uptake was not impaired.%%%%Neste trabalho foi analisada a influencia da fonte de carbono e do nivel de oxigenio sobre a localizacao da permease Lac12GFPp na levedura Kluyveromyces lactis. Inicialmente, foi mostrado que K. lactis apresenta afinidade maior para lactose do que para glicose. Os dados indicaram que a localizacao da permease Lac12GFPp dependente da KlSnf1p nao foi afetada pela presenca de glicose. A localizacao dessa permease foi similar quando K. lactis foi cultivada em galactose e glicose, mas diferente quando cultivada em lactose. Os niveis de mRNA do gene LAC12GFP expressos a partir do promotor ADH1, que nao esta sob a influencia da KlSnf1p, foram similares entre a linhagem selvagem e o mutante Klsnf1. Esse resultado mostra que a influencia da KlSnf1p sobre a expressao do gene LAC12GFP e exercida marcantemente somente em nivel pos-traducional. A analise da localizacao subcelular da permease de lactose no mutante Klsnf1 evidenciou que a permease e encontrada na membrana do vacuolo, sugerindo que Klsnf1p pode estar envolvida com a translocacao da permease para dentro do vacuolo. Em condicoes hipoxicas, ate 3 h de crescimento, foi observado aumento do sinal da permease de lactose na membrana…