THE EFFECT OF AMINO ACIDS ON THE UNFOLDED PROTEIN RESPONSE IN Saccharomyces cerevisiae

GULIAS JUAN FACUNDO; MUÑOZ, SEBASTIÁN ANIBAL; BIRENBAUM, JOAQUÍN MANUEL; BERMÚDEZ-MORETTI, MARIANA; CORREA-GARCÍA, SUSANA RAQUEL

Salta

Congreso; LV Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología molecular; 2019

SAIB

In eukaryotic cells, folding and modifications of membrane and secretory proteins take place in the endoplasmic reticulum (ER). When theprotein folding capacity of the ER is exceeded or experimentally impaired, unfolded proteins accumulate in the ER and activate the unfoldedprotein response (UPR). Amino acids are the building blocks of proteins and their levels are sensed by at least two evolutionarily conserved andintersecting mechanisms: one involving Gcn2 kinase and the other involving the TORC1 kinase. Gcn4, the main target of Gcn2, is a keytranscriptional activator of multiple amino acid biosynthetic genes, and Leu3 which acts mainly as a regulator of branched-chain amino acidmetabolism is itself under the control of Gcn4. In S. cerevisiae amino acids are also sensed by the sensor system SPS. The aim of this work wasto study how an excess of amino acids affects the UPR pathway and also to establish the signaling pathways involved. UPR was measured byusing a reporter gene assay in cells grown in the absence and in the presence of all amino acids. It is expected that an addition of amino acidsincreases the protein synthesis. We used wild type cells and cells deficient in the kinase Tor1, in the factors Gcn4 and Leu3, and in the essentialSPS protein Ssy1. We observed that both DTT and tunicamycin, two ER stressors, induce UPR to different extents, and that the presence ofamino acids triggers UPR except in cells lacking Ssy1 and Leu3. We also found that Leu3 is necessary for UPR. When ammonium is used asnitrogen source instead of proline, UPR activation is always observed. UPR response decreases in the presence of rapamycin, a TORC1inhibitor, in cells grown in both nitrogen sources independently on the presence of amino acids and is nearly abolished in tor1 cells. 136Tunicamycin has no effect on UPR in rapamycin treated cells. We analysed autophagy, a process that produces an increase in the intracellularamino acid pool. We observed that tor1 and leu3 cells present higher autophagy than wild type and that the addition of amino acids reduces theautophagic activity in all genotypes tested. In order to assess a link between UPR activation and chronological life span (CLS), we measuredCLS using the colony forming unit spot assay. Longevity was affected by growth conditions and genotypes assayed; however we could notestablish a direct correlation between UPR and CLS in all genotypes. Finally, we measured GCN4 expression as a marker of protein synthesisand we found that it decreases in cells lacking Tor1 and wild type cells treated with rapamycin whereas increases in leu3 cells. Altogether theseresults allow us to conclude that the addition of amino acids or the use of a rich nitrogen source activates UPR and that TORC1 and Leu3 arerequired in this process. We demonstrate that TORC1 and Leu3 are also involved in autophagy, amino acid biosynthesis and longevity but insome of these processes they act in an opposing manner.