Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae

Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cyst eine, a is any aliphatic residue, X is the carboxy terminal residue). To be tter understand the relationship between posttranslational processing and t he subcellular localization of Ras, a yeast genetic screen was undertaken b ased on the loss of function of a palmitoylation-dependent RAS2 allele, Mut ations were identified in an uncharacterized open reading frame (YLR246w) t hat we have designated ERF2 and a previously described suppressor of hypera ctive Pas, SHR5, ERF2 encodes a 41-kDa protein with four predicted transmem brane (TM) segments and a motif consisting of the amino acids Asp-His-His-C ys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD, Mutations w ithin the DHHC-CRD abolish Erf2 function. Subcellular fractionation and imm unolocalization experiments reveal that Era tagged with a triply iterated h emagglutinin epitope is an integral membrane protein that colocalizes with the yeast endo plasmic reticulum marker Kar2, Strains lacking ERF2 are viab le, but they have a synthetic growth defect in the absence of RAS2 and part ially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele, Ras2 proteins expressed in an erf2 Delta stra in have a reduced level of palmitoylation and are partially mislocalized to the vacuole, Based on these observations, we propose that Erf2 is a compon ent of a previously uncharacterized Ras subcellular localization pathway. P utative 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.