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

• Ano de defesa: 2007
• Autor(a) principal: Silveira, Wendel Batista da
• Orientador(a): Passos, Flávia Maria Lopes
• Banca de defesa: Costa, Maurício Dutra , Fietto, Luciano Gomes , Araújo, Pedro Soares de
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Kluyveromyces lactis; Permease de lactose; Metabolismo; Oxigênio
• Palavras-chave em Inglês: Kluyveromyces lactis; Lactose permease; Metabolism; Oxigen
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS
• Link de acesso: http://locus.ufv.br/handle/123456789/1527

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.