Analysis of the regulation of BAP2 in response to the quality of the nitrogen source

BALCAZAR DARIO; CARDILLO SABRINA; CORREA GARCÍA SUSANA; BERMÚDEZ MORETTI MARIANA

Vancouver

Congreso; Yeast Genetics and Molecular Biology Meeting; 2010

Saccharomyces cerevisiae branched amino acid permease Bap2 plays a mayor role in leucine, isoleucine and valine transport and its synthesis is regulated transcriptionally. BAP2 expression is induced by amino acids and it is not subject to NCR. Transcriptional activity from the BAP2 promoter is higher on an ammonium-based medium containing leucine than on a proline-based medium containing leucine. The SSY1 gene product is needed for the induced transcription of BAP2 in the presence of amino acids. Stp1 and Stp2, the two downstream effectors of SPS, activate BAP2 transcription by binding to the BAP2 promoter. Leu3 modifies BAP2 expression in cells grown on ammonium-based medium although the putative binding sites for Leu3 in BAP2 promoter do not seem to be essential for BAP2 induction. We found that the effect of amino acids, such as leucine or methionine, as inducers of BAP2 is stronger in a proline-based medium than in an ammonium-based medium. On this basis, our aim was to study the molecular mechanisms by which BAP2 transcription is regulated in cells grown using proline as the sole nitrogen source. BAP2 expression was estimated measuring galactosidase activity in cells carrying a BAP2-lacZ fusion gene. For western blots, strains transformed with a plasmid carrying a STP1-HA fusion gene were used. Chromatin immunoprecipitation assays were carried out using strains that express HA-Leu3 and HA-Stp1 fusion proteins.