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 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.

FIG. 1

Genetic screen to identify mutants that have an effect on the function of a palmitoylation-dependent RAS2 allele (ras2-ext). RJY1106 (MATa) and RJY1107 (MATα) cells were mutagenized as described in Materials and Methods. Mutants are defined by the inability to lose YCp52-Ras2 and are detected by the failure to form a sectored colony and 5-FOA sensitivity.

FIG. 2

Isolation and sequence analysis of ERF2. (A) B644 was identified from a low-copy-number yeast genomic library (ATCC 77162) by complementation of the 5-FOA sensitivity of an erf2-5 strain (RJY1081). The insert of B644 encompasses a 12.3-kb region of chromosome (Chrom) XII starting at nucleotide 617299 and ending at 629649. B644 was digested with the indicated restriction enzymes and religated, and a complementation test was performed. YLR246w was subcloned in B642 and shown to complement erf2-5. (B) Deduced amino acid sequence of ERF2 (YLR246w). The conserved CRD is shown in the black box, and predicted TM regions are underlined. Mutations isolated from the ERF screen are shown in italic, and site-directed mutations are italicized and boldface. Nonsense mutations uncovered in the screen are indicated with asterisks. (C) Sequence alignment of the five putative S. cerevisiae ERF2 orthologs and a representative list of possible CRD homologs from other organisms: S. pombe (accession no. CAA20305), A. thaliana (CAA23000), Drosophila (AAD34351), C. elegans (CAA21738), M. muscus (AI322485), and H. sapiens (AA112746). Amino acid identities and similarities to Erf2 are shaded black and gray, respectively.

FIG. 3

Erf2 is an integral membrane protein. (A) Kyte-Doolittle analysis of hydrophobicity predicts the existence of four TM segments. (B) Membrane fractionation of Erf2-HA₃. Total cell lysates were prepared from RJY1318 harboring B754 (Erf2-HA3) as described in Materials and Methods. The extracts were then treated with buffer, 0.6 M NaCl, 1.6 M urea, 0.1 M Na₂CO₃ (pH 11), 1% Triton X-100, 1% sodium cholate, 1% Triton X-100 and 1% sodium cholate (Triton + NC), or 1% SDS. Following incubation on ice for 30 min the extracts were separated into cytosolic (S100) and crude membrane (P100) fractions. Samples were resolved by SDS-PAGE and visualized by immunoblotting with anti-HA monoclonal antibody 12CA5 (BAbCo) or anti-Ras monoclonal antibody Y13-259. Immunocomplexes were visualized by using an HRP-conjugated secondary antibody and enhanced chemiluminescence (Pierce SUPER-SIGNAL).

FIG. 4

Immunolocalization of Erf2-HA₃. (A) RJY1318 harboring B745 (Erf2-HA₃) was grown and prepared for immunofluorescence as described in Materials and Methods. Erf2-HA₃ was visualized with anti-HA monoclonal antibody 12CA5 (BAbCo) diluted 1/400 followed by Oregon Green 488-conjugated IgG (1:240) (a to c). Kar2 was visualized with an affinity-purified anti-Kar2 antibody (1:2,000) followed by lissamine rhodamine-conjugated anti-rabbit IgG (1:240) (d to f). DAPI staining of the nuclei is also shown (g to i). Cells were examined in a Zeiss Axioskop microscope. (B) P13 and P100 fractionation of RJY1318 harboring B754 (Erf2-HA₃). Samples were resolved by SDS-PAGE and visualized by immunoblotting with an anti-HA (12CA5; BAbCo), anti-Dpm1, anti-Kar2, or anti-Pma1 antibody as described in Materials and Methods. Immunocomplexes were visualized as in Fig. 3. PM, plasma membrane.

FIG. 5

ras2Δ erf2Δ strains grow slowly. (A) Heterozygous diploids RJY1412 (top) and RJY1301 (middle) were sporulated, and a representative tetrad is shown. The bottom panel shows tetrads resulting from sporulation of RJY1301 harboring the MET25-RAS1 plasmid (B804). Colonies were grown on rich medium containing glucose (YEPD) for 3 days. (B) Indicated strains were streaked onto YEPD medium and shown to have a severe growth phenotype.

FIG. 6

In vivo labeling with [³H]palmitate. Strains RJY1270 wild type [WT]), RJY1272 (erf2Δ), and RJY1274 (erf4/shr5Δ) were labeled with [³H]palmitate as described in Materials and Methods. (A) GST-Ras2 was purified by glutathione affinity chromatography and resolved by SDS-PAGE, and the gel was subjected to fluorography (top) or anti-GST immunoblotting (bottom). (B) Total lysates from [³H]palmitate-labeled RJY1270 (WT) and RJY1272 (erf2Δ) were resolved by SDS-PAGE and subjected to fluorography (top) or anti-GST immunoblotting (bottom). The migration positions of prestained molecular weight markers (Gibco-BRL) are indicated on the left in kilodaltons.

FIG. 7

Crude membrane association of Ras2 and Ras2-ext in ERF2 and erf2Δ strains. Total cell lysates were prepared from RJY1107 (ERF2) or RJY1277 (erf2Δ) as described in Materials and Methods. The extracts were separated into S100 and P100 fractions. Ras2 and Ras2-ext were visualized by immunoblotting using a rat anti-ras monoclonal antibody (Y13-259) and anti-rat HRP-conjugated secondary antibody (Amersham). Immunocomplexes were visualized as in Fig. 3.

FIG. 8

Subcellular localization of palmitoylated and nonpalmitoyled GFP-Ras proteins in ERF2 and erf2Δ strains. (A) RJY266 (ERF2) and RJY1318 (erf2Δ) were transformed with GFP-Ras2 (B701) (A) or GFP-Ras2(SCIIS) (B763) (B), and cells were grown in SC medium lacking uracil (4% galactose) to an optical density at 660 nm of 1.0. Vacuoles were visualized by staining with FM4-64. Cells were visualized directly by confocal microscopy (Bio-Rad MRC1024) using a 60× objective.

FIG. 9

Heat shock sensitivity of ERF2 and erf2Δ strains. ERF2 (+; LRB759) or erf2Δ (Δ; RJY1438) strains were transformed with pRS315 plasmids expressing Ras2(CCIIS) (B250), Ras2(V19,CCIIS) (B561), or Ras2(V19,SCIIS) (B562). The strains were subjected to heat shock for 10 min or 30 min and a serial dilution of cells was plated on SC medium plates lacking leucine as described in Materials and Methods.