Neo1p is an essential yeast member of the highly conserved Drs2 family of P-type ATPases with proposed aminophospholipid translocase activity. Here we present evidence that Neo1p localizes to endosomes and Golgi elements. In agreement with that finding, the temperature-sensitive neo1-37 and neo1-69 mutants exhibit defects in receptor-mediated endocytosis, vacuole biogenesis, and vacuolar protein sorting. Furthermore, neo1 mutants accumulate aberrantly shaped membranous structures most likely derived from vacuoles and the endosomal/Golgi system. At permissive temperatures, HA-Neo1-69p, like wild-type Neo1p, is stable and associates with endosomes. In contrast, HA-Neo1-37p is rapidly degraded and is predominantly retained within the endoplasmic reticulum (ER). Thus, the two neo1 alleles affect the stability and localization of the mutant polypeptides in different ways. A C-terminally truncated and a C-terminally epitope-tagged version of Neo1p are nonfunctional and also mislocalize to the ER. In agreement with a role within the endomembrane system, Neo1p exhibits genetic and physical interactions with Ysl2p, a potential guanine nucleotide exchange factor for Arl1p. Interestingly, deletion of ARL1 rescues the temperature sensitivity of neo1-37 and neo1-69. We demonstrate that Arl1p in its myristoylated and GTP-bound form is responsible for the inhibitory effect. Thus, Neo1p, Ysl2p, and Arl1p represent three proteins that collaborate in membrane trafficking within the endosomal/Golgi system.
Copyright 2004 American Society for Microbiology