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 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 erf2Delta 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.     

c-Myc localization within the nucleus: evidence for association with the PML nuclear body

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.     

Determination of copy number of c-Myc protein per cell by quantitative Western blotting

The protooncogene c-Myc plays a key role in growth control, differentiation, and apoptosis. An abnormally high expression of c-myc has been found to be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level. Few studies have been published on quantitative Myc protein determination. A major drawback of ELISA (enzyme-linked immunosorbent assay) methods is the uncertainty of the specificity of the antibody reaction. In contrast, antibody specificity can be easily controlled by Western/immunoblotting. Here we describe a method to quantify c-Myc protein in primary human IMR90 lung fibroblasts based on Western blotting. Using a high-resolution polyacrylamide gel, we were able to differentiate the cellular c-Myc protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined both the total c-Myc protein content per cell and its distribution in the cytoplasmic and nuclear fractions. About 4000 c-Myc protein molecules were detected in the cytoplasmic fraction and 29,000 copies in the nuclear fraction for proliferating human lung fibroblasts IMR90. The ratio of nuclear (active) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferating cells to 2.5:1 for confluent cells.     

Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae

Most Ras proteins are posttranslationally modified by a palmitoyl lipid moiety through a thioester linkage. However, the mechanism by which this occurs is not known. Here, evidence is presented that the Ras2 protein of Saccharomyces cerevisiae is palmitoylated by a Ras protein acyltransferase (Ras PAT) encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the membrane of the endoplasmic reticulum and contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Erf2p co-purifies with Erf4p (26 kDa) when it is expressed in yeast or in Escherichia coli. The Erf2p/Erf4p complex is required for Ras PAT activity, and mutations within conserved residues (Cys(189), His(201), and Cys(203)) of the Erf2p DHHC-CRD domain abolish Ras PAT activity. Furthermore, a palmitoyl-Erf2p intermediate is detected suggesting that Erf2p is directly involved in palmitate transfer. ERF2 and ERF4 are the first genes identified that encode a palmitoyltransferase for a Ras GTPase.