Mutational Analysis of Saccharomyces cerevisiae Erf2 Reveals a Two-step Reaction Mechanism for Protein Palmitoylation by DHHC Enzymes*

Abstract

DHHC protein acyltransferases (PATs) catalyze the palmitoylation of eukaryotic proteins through an enzymatic mechanism that remains largely unexplored. In this study we have combined genetic and biochemical approaches to examine the molecular mechanism of palmitate transfer of the yeast Ras PAT, which is composed of Erf2 and Erf4. The palmitoylation reaction consists of two steps; they are autopalmitoylation of the enzyme to create a palmitoyl-Erf2 intermediate followed by the transfer of the palmitoyl moiety to the Ras substrate. Palmitoyl-CoA serves as the palmitate donor. To elucidate the kinetic properties of the Erf2⅐Erf4 PAT, we have developed a coupled enzyme assay that monitors the turnover of the palmitoyl-enzyme species indirectly by measuring the rate of CoASH release. Mutational analysis indicates that the DHHC motif constitutes the catalytic core of the enzyme required for autopalmitoylation and palmitoyl transfer to the Ras2 substrate. In the absence of Ras2, the palmitoyl-Erf2⅐Erf4 complex undergoes a cycle of hydrolysis and repalmitoylation, implying that in the presence of palmitoyl-CoA, the complex is autopalmitoylated and competent to transfer palmitate to a protein substrate.Post-translational modifications extend the functional and regulatory repertoire of proteins. In the case of protein lipidation, the addition of prenyl, palmitoyl, myristoyl, and cholesterol moieties affects subcellular localization and protein trafficking, solubility, and degradation (1, 2). Protein S-palmitoylation is the reversible thiol esterification of cysteine residues that is catalyzed by a family of DHHC motif-containing proteins called protein acyltransferases (PATs) 2 or simply palmitoyl transferases (3-5). Protein palmitoylation was first described over 30 years ago and has been implicated in key cellular processes including cell growth and proliferation, protein trafficking, protein turnover, and vesicle fusion (6, 7). The reversible nature of protein palmitoylation makes it well suited to play a regulatory role, including in heterotrimeric and small GTP-binding proteins, receptors, and ion channels (8, 9). PAT enzymes share a number of common features including four transmembrane domains and a canonical Asp-His-His-Cys sequence (DHHC motif) that has been hypothesized to be the catalytic center of the enzyme (5, 7, 10). Despite the central importance of protein palmitoylation in many physiological processes, the molecular mechanism of the palmitoylation reaction remains poorly understood.There are 7 DHHC PATs in yeast and 23 in mammals (7). Based on in silico topology (TMPRED) computer simulations, the peptide loop containing the signature DHHC residues is predicted to immediately precede a transmembrane domain placing the DHHC motif in juxtaposition to the membrane (11). In addition to the DHHC motif and the transmembrane domains, most but not all PATs contain a cysteine-rich domain (CRD) (7). The exceptions include Akr1, Akr2, Pfa5 (yeast), and DHHC22 (mammals), which lack one or more of the conserved cyste...

Mutations in the X-linked Intellectual Disability Gene, zDHHC9, Alter Autopalmitoylation Activity by Distinct Mechanisms

“…Two genetic variants of zDHHC9, R148W, and P150S, fall within the (R/K)XR motif and are asso-ciated with the Marfanoid features and psychological disorders characteristic of syndromic XLIDs. In a recent study, we screened the DHHC CRD of yeast Erf2 and found that the RPPR motif is involved in the catalytic step after the initial formation of the Erf2-palmitoyl thioester bond intermediate (15). This observation suggests that the palmitoylation defect in the XLID alleles of zDHHC9 might alter the kinetics of the autopalmitoylation step of the PAT reaction.…”

“…The resulting extract was spun at 3000 ϫ g for 15 min to remove cellular debris and unbroken cells to yield a whole cell extract. Purification procedure of the zDHHC9-GCP16 enzyme complexes from the whole cell extract has been previously described for Erf2-Erf4 (15).…”

“…Coupled PAT Assay-The coupled PAT assay has been described previously (15). The 200-l reaction contained 2 mM 2-oxoglutarate (␣-ketoglutamic acid), 0.25 mM NAD ϩ , 0.2 mM thiamine pyrophosphate, 2 g of purified zDHHC9-GCP16 or zDHHC9-GCP16 mutant complex, 1 mM EDTA, 1 mM dithiothreitol, 32 milliunits of 2-oxoglutarate dehydrogenase (␣-ketoglutarate dehydrogenase), in 50 mM sodium phosphate, pH 7.2.…”

“…In the second step, palmitate is transferred to the thiol of a cysteine of the protein substrate. In the absence of protein substrate, the thioester is hydrolyzed, releasing free palmitate and enzyme poised to react with another molecule of palmitoylCoA (15). In the Saccharomyces cerevisiae Erf2-Erf4 Ras PAT, the rate of the autopalmitoylation/hydrolysis cycle in the absence of Ras substrate is regulated by the Erf4 accessory subunit, although the molecular mechanism has not been elucidated (16).…”

“…The zDHHC-catalyzed palmitoylation reaction utilizes a two-step mechanism. In the first step, a substrate-enzyme (PAT) intermediate is formed via a thioester linkage between palmitate from palmitoyl-CoA and the cysteine of the DHHC motif, a process referred to as autopalmitoylation (15). The proposed juxtaposition of the PAT catalytic active site with the cytosolic surface of the membrane implies that the enzyme can utilize membrane-associated palmitoylCoA as the acyl donor and suggests that after the formation of the palmitoyl-PAT intermediate, the active site of the enzyme will be tethered to the membrane through the palmitoyl moiety.…”