A DEFICIENCY IN UGA3 PRODUCED CHANGES IN THE PROTEOME THAT ALTERED INTRACELLULAR AMINO ACID CONTENT

CORREA GARCIA SUSANA; MUÑOZ, SEBASTIÁN ANÍBAL; BERMUDEZ MORETTI MARIANA; GULÍAS, JUAN FACUNDO

Congreso; SAIB; 2020

SAIB

The yeast S. cerevisiae is able to use a wide variety of compounds as nitrogen source. Moreover, it is also able to synthesize all L-amino acids used in protein synthesis. The non-proteinogenic amino acid GABA can be used as sole nitrogen source and the transcription factor Uga3 was previously described as a transcription factor specific of the GABA utilization pathway. Together with the pleiotropic transcription factor Dal81, Uga3 regulates the three UGA genes in a positive GABA-dependent way. However, in earlier reports we showed evidence of new functions of Uga3 such as its role in the regulation of BAP2 gene, that encodes one of the permeases of branched amino acids. Moreover, we found that Uga3, but not the whole GABA shunt, is associated to both thermal and oxidative stress tolerance. Looking forward to find new functions of the transcription factor Uga3, we compared the proteome of uga3 mutant cells and wild type cells. Using label-free quantitative mass spectrometry we detected 67 proteins differentially expressed, fifty of them were sub-represented in the mutant while only seventeen were over-represented in the absence of Uga3. After analysis in silico, using PANTHER, DAVID and STRING data bases, we found that eight of these proteins, ARG5,6, DAL5, GDH2, ALT1, LYS2, VBA1, GAP1, GLT1, are related to amino acids metabolism, either in biosynthesis, catabolism or transport through the plasma membrane. We measured expression of these genes using RT-qPCR in wild type and uga3∆ cells and we found that the changes in protein levels observed for Vba1, Arg5,6 and Gdh2 correlated with changes in VBA1, ARG5,6 and GDH2 mRNA. Moreover, we found that the well documented arginine-repression of ARG5,6 depended on Uga3. Finally, to study the outcome of the changes produced by the absence of Uga3, we compared the intracellular amino acids content in uga3∆ and wild type cells using coupled NMR-HPLC. Our preliminary results showed changes in the accumulation of eight amino acids. These changes could be the cause, at least in part, of the high stress sensitivity observed in the uga3∆ mutant strain.