ERF4, a subunit of the yeast Ras-specific palmitoyltransferase, is required for plasma membrane localization of Ras in Saccharomyces cerevisiae

Plasma membrane localization is crucial for the functionality of Ras proteins. This is dependent on posttranslational modifications, especially farnesylation and palmitoylation. While farnesyltransferase was characterized years ago, the proteins responsible for the palmitoylation of Ras had eluded research efforts until recently. In a genetic screen aimed at identifying proteins that have an effect on Ras function, two genes were recovered, ERF2 and ERF4. Recent studies have revealed the roles of Erf2p in Ras palmitoylation and plasma membrane localization. Deletion of ERF2 incurs severe reduction of Ras palmitoylation in vivo and partial mistargeting of Ras to vacuole membrane. ERF4 deletion displays the same phenotype. Deletion of both genes has no additive effect. In contrast, Ras is completely depleted from the plasma membrane once the classical secretory pathway is blocked in addition to the null mutation in ERF2 or ERF4. These lines of evidence lead to the speculation that Erf2p and Erf4p function in the same pathway that is independent of the classical secretory pathway. To test this hypothesis, I embarked on a genetic high copy suppression assay first. An allele-specific interaction between ERF2 and ERF4 was observed. Further characterization of Erf4p and its interaction with Erf2p strongly suggests that these proteins form a complex that is localized to endoplasmic reticulum. At approximately the same time, Erf2p was shown to exhibit Ras-specific palmitoyl acyl transferase (Ras PAT) activity, in the presence of Erf4p. Through mutagenesis and biochemical studies, I found that Erf4p is also an indispensable component of the Ras PAT. In addition, Erf2p is phosphorylated in the presence of a functional Erf4p. The role of this phosphorylation is not clear. That Erf4p is a component of the Ras PAT is somewhat unexpected, because this protein is not well conserved, unlike Erf2p, the other protein in this complex. Many Erf2p homologs have been identified, and multiple alignments and phylogenetic studies were performed to categorize these proteins. The yeast Erf2p paralogs were studied and at lease two of these proteins show weak PAT activity. In summary, this work establishes that Erf4p is a crucial component of the Ras PAT and is required for Ras targeting to the plasma membrane.