Palmitate modifies both peripheral and integral membrane proteins and its addition can be permanent or transient, which makes it unique among the lipid modifications of proteins. The presence of palmitate on a protein affects how the protein interacts with lipids and proteins in a membrane compartment, and the reversibility of palmitoylation allows different modes of trafficking between membrane compartments. Here, we review recent studies that have provided insights into the mechanisms that mediate the functional consequences of this versatile modification.

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 Dozens of cellular and viral proteins are posttranslationally modified with palmitate or other long-chain fatty acids through a reversible thioester linkage (1, 2). Examples are cell surface receptors, viral glycoproteins, and signal transducers including Ras, heterotrimeric G proteins, and nonreceptor tyrosine kinases. Palmitoylation is almost exclusively a property of membrane proteins and can occur on intracellular membranes early in the secretory pathway or at the plasma membrane. Although this modification was first described over 30 years ago, the molecular mechanism of palmitate addition has not been elucidated and has been a matter of controversy. A palmitoyltransferase activity assayed using mammalian H-Ras as a substrate was purified and identified as thiolase A, an enzyme required for fatty acid ␤-oxidation (3, 4). The localization of this enzyme in peroxisomes makes it an unlikely candidate for a physiological regulator of Ras palmitoylation. Palmitoyltransferase activities assayed using viral glycoproteins, the nonreceptor tyrosine kinase p59 fyn , or G-protein heterotrimer as substrates have been detergent-solubilized, but the instability of the activity has hampered purification and molecular identification (5-7). A candidate palmitoyltransferase for Drosophila hedgehog was recently identified as skinny hedgehog/sightless (5, 6). Hedgehog is modified by cholesterol at the C terminus and palmitoylated through an atypical cysteine amide linkage at the N terminus. The failure to identify a palmitoyltransferase that acylates through a conventional thioester linkage, coupled with the observation that spontaneous and efficient transfer of fatty acid from acyl-CoA to proteins occurs in vitro, has lead to the suggestion that proteins autoacylate in vivo (7-9).Plasma membrane localization of Ras requires farnesylation of the CaaX box cysteine via a thioether linkage, -aaX proteolysis, and carboxylmethylation (see reviews in Refs. 10, 11). With the exception of K-Ras-4b, human Ras proteins are also palmitoylated on one or more neighboring cysteines via a thioester linkage. Palmitoylation is required for efficient plasma membrane localization and transforming activity of oncogenic forms of Ras (12). Previously, we described palmitoylationdependent alleles of yeast RAS2 and a genetic screen designed to identify mutations that render cells inviable if Ras2p is not palmitoylated (13,14). Mutations in two genes, ERF2 and ERF4/SHR5, were identified that resulted in diminished palmitoylation of Ras2p and mislocalization of 15). Erf2p is a 41...

Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H-and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 codistribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2⅐Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9⅐GCP16 exhibits substrate specificity, palmitoylating H-and N-Ras but not myristoylated G ␣i1 or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9⅐GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the a-carboxyl group is also methyl esterified. To determine if ras

A rapid and convenient method of protein purification involves creating a fusion protein with glutathione S-transferase (GST) (Smith and Johnson, Gene 67, 31-40, 1988). In this report, we describe two vectors for the conditional expression of GST fusions in Saccharomyces cerevisiae. The parent plasmid is based on a high-copy, galactose-inducible shuttle vector previously described (Baldari et al., EMBO J. 6, 229-243, 1987). We have demonstrated the use of this system by creating fusions between GST and the yeast RAS2 gene. GST-Ras2 fusion proteins undergo the post-translational modifications required for Ras2p to become membrane localized. These vectors provide a useful system for the expression and purification of eukaryotic proteins requiring post-translational modification.