STUDIES ON MECHANISMS UNDERLYING REPLICATIVE LIFESPAN EXTENTION IN YEAST WITH DISASSEMBLED EISOSOMES MEMBRANE DOMAINS

SALZMAN, VALENTINA; AGUILAR, PABLO S.; NIEVAS, MICAELA; CORREA TEDESCO, FRANCISCO

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB

Eisosomes are plasma membrane (PM) domains concentrating lipids and proteins Saccharomyces cerevisiae. Deletion of structural protein PIL1 leads to disappearance of these domains, very few and largeclusters remain. Performing RLS (replicative lifespan) assays we found that a knockout strain for PIL1 has significantly enhanced longevity. We are interested in determining the mechanism underlying this phenotype.Eisossomes concentrate at least 25 different proteins including signalling proteins and nutrient transporters such as Tat2, a permease that mediates high-affinity tryptophan (Trp) import. Knowing that TAT2 gene deletioncauses nutrient limitation and RLS extension in S. cerevisiae, we challenged a specific hypothesis: eisosomes´ disassembly decreases Tat2 permease activity, increasing lifespan. We set up an assay to measure 3HTrp importin vivo to compare Trp uptake between pil1 and WT strains. In addition to Tat2, there is a second protein (Tat1) that transports Trp with lesser affinity. We found that at [Trp] lower than 1uM, most of the amino acid is importedthrough Tat2 in the WT strain. Kinetic parameters for Trp uptake in the WT strain were estimated, we found that Kmap value is 30 uM..It was not possible to infer the contribution of Trp permeases individually at highersubstrate concentrations, therephoreTat1 low affinity permease gene was eliminated and Trp uptake was compared between tat1 and tat1pil1 strains. We found that eisosomes´ disassembly increasesTat2 permease activity,therephore Tat2 is not responsible for pil1 mutant RLS extention phenotype.