THE ROLES OF TWO SMALL GTP-BINDING PROTEINS ARL1 AND YPT6 IN AUTOPHAGY
Rosenwlad, Anne G.
Autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membraned structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow for hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that two monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy at 37°C. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at 37°C, although autophagy proceeds normally at the normal growth temperature, 30°C. Specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these two proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at 37°C. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein complex (GARP) to the PAS to regulate the anterograde trafficking of Atg9. We also carried out a high copy number suppressor screen to identify genes that when overexpressed suppress the rapamycin-sensitive phenotype of arl1Δ and ypt6Δ strains at 37°C. From the screen results, we selected COG4, SNX4, TAX4, IVY1, PEP3, SLT2 and ATG5, either membrane traffic or autophagy regulators, to further test whether they can suppress the autophagy defects of arl1Δ and ypt6Δ strains. We identified COG4, SNX4 and TAX4 to be the suppressors specifically for the arl1Δ strain; IVY1 and ATG5 for the ypt6Δ strain. In conclusion, through this work we show two membrane traffic regulators, Arl1 and Ypt6, have novel roles in autophagy.
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