Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

Abstract

Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal C aaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependent RAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele. Ras2 proteins expressed in an erf2Δ strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.

ACKNOWLEDGMENTS

We thank Mark Rose (Princeton) and Tom Stevens (Oregon) for antibodies, Jasper Rine (Berkeley) for the GFP-Ras plasmid, and Lucy Robinson for strains. In addition, we thank Lois Weisman and Jan Fassler for many helpful discussions and for reading the manuscript. Immunolocalization studies were done with the assistance of Cherie Malone and of Tom Monniger, University of Iowa Central Microscopy Research Facility. FM4-64 labeling was done with the skillful assistance of Cecilia Bonangelino of the Weisman lab. We especially thank members of the lab (Cherie Malone, Hong Lin, Addison Ault, and Lihong Zhao) for their input throughout the study.

This work was supported by grant CA50211 from the National Cancer Institute.