The Erf4 subunit of the yeast Ras palmitoyl acyltransferase is required for stability of the Acyl-Erf2 intermediate and palmitoyl transfer to a Ras2 substrate

Abstract

Protein S-palmitoylation is a posttranslational modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a reversible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and trafficking, protein aggregation, and protein degradation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the subcellular trafficking of Ras family GTPases. The Ras protein acyltransferase (PAT) consists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respectively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-mediated pathway. In addition, Erf4 is required for the stable formation of the palmitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermediate is increased, resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.

FIGURE 1.

Erf2 is destabilized in the absence of Erf4. Strain RJY1620 expressing 13xMyc:ERF2 and B1414 (ERF4) or 13xMyc:ERF2 alone was grown to an A₆₀₀ of 1.0 and treated with cycloheximide (25 μg/ml) to inhibit protein translation, and samples were removed at the indicated times after cycloheximide addition. A, shown is a representative Western analysis of ERF2 ERF4-expressing strains (i) or ERF2 erf4Δ strains (ii) probed with antibodies to the c-Myc epitope that tags Erf2. The blots were also probed with antibodies to phosphoglycerate kinase (PGK) as a lane-loading control. B, shown is a semi-log graphical representation of the Western blot shown in A after densitometry (ImageJ, NIH) to empirically determine the half-life of Erf2 in the presence of Erf4 (153 min) and in the absence of Erf4 (25 min). C, shown is a comparison of half-lives of Erf2 in the presence of Erf4 and Erf2, Erf2ΔN, and Erf2ΔC in the absence of Erf4. The bar graph shows the data from two independent experiments (black and gray bars) determining the half-life of Erf2.

FIGURE 2.

Degradation of Erf2 involves polyubiquitinylation and the ERAD system. A, upper panel, to determine if Erf2 undergoes ubiquitinylation, extracts from diploid erf4Δ strains expressing FLAG:ERF2 with and without pUb221 (6×His:ubiquitin) or FLAG:ERF2–6R with and without pUb221 were treated with 6 m guanidine-hydrochloride (to denature and dissociate all non-covalently associated proteins), and ubiquitinylated proteins were isolated using Ni-NTA-agarose, separated by SDS-PAGE, and immunoblotted with ant-FLAG antibodies (to detect Erf2). Strains expressing both plasmids produced polyubiquitin-conjugated Erf2 molecules as seen by a smear larger than the apparent molecular weight of FLAG:Erf2. Lower panel, whole cell extracts from the strains used in the upper panel were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody to demonstrate the presence of FLAG:Erf2. B, the half-life of 13xMyc:Erf2 in the absence of Erf4 is increased by deleting the ER quality control components. The bar graph shows the data from two independent experiments (black and gray bars) for the half-life of Erf2 in isogenic strains erf4, yos9 erf4, rpn10 erf4, hrd1 erf4, ubc7 erf4, and doa10 erf4 compared with the wild type (ERF4) strain.

FIGURE 3.

The C-terminal 58 amino acids of Erf2 are sufficient to promote degradation. A, shown is a schematic representation of ER localized acyltransferase, Pfa4, with the C-terminal FLAG epitope and Erf2 58 amino acid additions. The amino acid sequence below the schematic compares the wild type Erf2 C-terminal 58 amino acids with that of Erf2–6R in which the six lysines are mutated to arginines (asterisks). B, representative Western analysis comparing the amounts of Pfa4:FLAG, Pfa4:FLAG:Erf2C58, Pfa4:FLAG:Erf2C58–6R, FLAG:Erf2, and FLAG:Erf2–6R at the indicated times after cycloheximide (25 μg/ml) addition probed with antibodies to the FLAG epitope (Sigma). C, shown is a comparison of the half-lives of Pfa4:FLAG, Pfa4:FLAG:Erf2(C58), Pfa4:FLAG:Erf2(C58–6R), FLAG:Erf2, and FLAG:Erf2–6R. The bar graph shows the data from two independent experiments (black and gray bars) determining the half-life of the indicated fusion proteins.

FIGURE 4.

Stabilized Erf2 cannot suppress the loss of ERF4. Shown is a functional plasmid shuffle assay to determine the ability different Erf2 stabilizing conditions to suppress the loss of ERF4. A, the genes for ERAD components were deleted from RJY1620 and plated on synthetic medium lacking uracil to demonstrate the presence of the sectoring plasmid. These strains, harboring pRS314 (TRP1) or B1414 (pRS314ERF4), were replicated to synthetic medium lacking tryptophan and supplemented with 5′-FOA to select for those strains capable of losing the URA3 linked RAS2 episome. B, shown is a series dilution of strain RJY1888 cultures harboring plasmids expressing the ERF2 alleles with and without ERF4. Cells were spotted in 10-fold dilutions (10⁴ initial cfu) on medium lacking leucine (left panel) and medium lacking leucine supplemented with 5′-FOA (right panel) and incubated for 4 days at 30 °C.

FIGURE 5.

Erf4 dependence of Erf2 autopalmitoylation. A, in vitro autopalmitoylation reactions using Bodipy C12:0-CoA as the acyl donor are shown. Top panel, in vitro autopalmitoylation reactions were separated using SDS-PAGE under non-reducing conditions, and the fluorescence was visualized using excitation 488-nm/emission 520-nm filters (Typhoon, GE). The middle panel of A shows a representative Western blot (WB) analysis used to quantify the amount of Erf2 and Erf2 mutants. The bar graph shows the amount of autopalmitoylation normalized to the amount of Erf2 protein present in each sample (bottom panel). The asterisk denotes cross-reactivity of anti-FLAG with reduced small chain IgG from the antibody coated agarose beads. B, shown is post-steady state autopalmitoylation and hydrolysis fluorescence assay that couples the production of CoASH, a product of the autopalmitoylation reaction, with the reduction of NAD⁺ (NADH) using α-ketoglutarate dehydrogenase. Assays were performed varying the amount of palmitoyl-CoA. The background values were determined by performing the assays using the analogous catalytically impaired Erf2 enzymes (Erf2 C203S), and those values were subtracted from the values obtained using the active Erf2 enzymes. The data represent Erf2ΔC (open triangle), Erf2ΔC-Erf4 (closed triangle), Erf2-Erf4 (open circle), Erf2 (closed circle), Erf2–6R-Erf4 (open square), and Erf2–6R (closed square). The data were fit using Prizm software, n = 4. K_m, V_MAX, and k_cat/K_m values are shown in Table 3.

FIGURE 6.

Erf2 requires Erf4 to transfer palmitate to Ras2. In vitro transfer reactions using Bodipy C12:0-CoA as the acyl donor. First panel, the products of the in vitro acyl transfer reactions were separated using SDS-PAGE under non-reducing conditions. MBP:mCherry:Ras2CT35 (100 pmol) was used as the protein substrate. The amount of Bodipy C12:0 transfer was determined by scanning the gel using excitation 488-nm/emission 520-nm filters (Typhoon, GE Healthcare). Second panel, the amount of MBP:mCherry:Ras2CT35 is identical in all cases as determined by measuring the fluorescence of the mCherry chromophore using excitation 520-nm/emission 610-nm filters (Typhoon). Third panel, the same enzyme preparations used to determine the autopalmitoylation activity (Fig. 5A) were also used for determining the acyl transfer activity and were quantified by Western blot analysis using antibodies to the FLAG epitope. The asterisk denotes cross-reactivity of anti-FLAG with reduced small chain IgG from the antibody-coated agarose beads. The bar graph below shows the amount of Bodipy C12:0 transferred relative to the amount of MBP:mCherry:Ras2CT35:Ras2 substrate used in the reaction (pmol/min/μmol of MBP:mCherry:Ras2CT35) normalized to the amount of Erf2 protein (Fig. 5A, bottom panel) present in each sample. The background fluorescence of the MBP:mCherry:Ras2CT35 alone lane (no enzyme) was subtracted from the sample lanes.