Abstract
Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3Delta crf1Delta background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1Delta mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p-Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.