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

CORREA, FRANCISCO; NIEVAS, MICAELA; SALZMAN, VALENTINA; AGUILAR, PABLO

SALTA

Congreso; 55th Annual Meeting Argentine Society of Biochemetry and Molecular Biology; 2019

SAIB

Eisosomes are plasma membrane (PM) domains concentrating lipids and proteins in the budding yeast Saccharomyces cerevisiae. These are domains shaped as 200?400-nm long and 50-nm-deep invaginated furrows structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Deletion of PIL1 leads to disappearance of invaginations, very few and large clusters persist at the PM concentrating a fraction of the original eisosomal proteins. We study eisosomes´ role in aging measuring the number of daughters produced by yeast mother cells of a dividing culture (replicative aging model, RLS). Performing RLS assays we found that knockout strains for PIL1 have significantly enhanced longevity. We are interested in determining the mechanism underlying this phenotype. Eisosomes have been associated with the regulation of endocytosis of certain eisosomal proteins as well as the functionality of some of them. These domains 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 deletion causes 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 import in vivo in order to compare Trp uptake between pil1 mutant and WT strain. In addition to Tat2, there is a second protein (Tat1) that transports Trp with lesser efficiency. Using tat2tat1 and tat2 mutants as control strains, we found that at [Trp] lower than 1uM, most of the amino acid is imported through Tat2 in the WT strain. Under these conditions, PIL1 deletion does not affect Trp uptake. Next, we estimated the kinetic parameters for Trp uptake in the WT strain and found that apparent Km (Kmap) value is 30 uM. Taking into account that Tat2 activity could be lower in a strain with disassembled eisosomes not only because Kmap is affected but also because Vmax could be reduced, we decided to extend the range of [Trp] tested when comparing Tat2 import activity. As the kinetic parameters of both Trp permeases individually are unknown, it was not possible to infer the contribution of each of them at high substrate concentrations. Therefore, Tat1 low affinity permease gene was eliminated and Tat2 activity was measured in a wide range of substrate concentrations in tat1 and tat1pil1 strains. Additionally, to determine if eisosomes disassemble decreases Tat2 protein levels; we imaged cells expressing Tat2?GFP by fluorescence microscopy and quantified the protein fraction localized at the PM. The results contribute to understanding Tat2 role in pil1 mutant longevity phenotype in order to further describing complex S. cerevisiae aging process and nanoscale PM domains function.