Purification and Biochemical Characterization of a Protein-palmitoyl Acyltransferase from Human Erythrocytes

Abstract

Protein palmitoylation involves the post-translational attachment of palmitate in thioester linkage to cysteine residues of proteins. The labile nature of the thioester linkage makes possible the palmitoylation-depalmitoylation cycles that have emerged in recent times as additions to the repertoire of cellular control mechanisms. However, detailed understanding of these cycles has been limited by the lack of knowledge of the transferases and thioesterases likely to be involved. Here, we describe the purification of a protein-palmitoyl acyltransferase (PAT) from human erythrocytes. PAT behaved as a peripheral membrane protein and catalyzed the attachment of palmitate in thioester linkage to the ␤-subunit of spectrin. On SDS-polyacrylamide gel electrophoresis, PAT appeared as a 70-kDa polypeptide. Antibody against this polypeptide could immunodeplete PAT activity from the crude extract, confirming the assignment of the 70-kDa polypeptide as PAT. PAT-mediated spectrin palmitoylation could be inhibited by nonradioactive palmitoyl-, myristoyl-, or stearoyl-CoA. The apparent K m for palmitoyl-CoA was 16 M.A large number of proteins in cells are modified by the covalent attachment of long chain saturated fatty acid residues (1-3). The two most common modifications involve acylation with myristate and palmitate. Myristate is usually attached to an N-terminal glycine via an amide bond in a relatively stable linkage. Palmitate is usually attached via a thioester bond to cysteine residues of proteins. Palmitoylation occurs in membrane proteins with hydrophobic transmembrane segments as well as in hydrophilic proteins such as Ras (1-4). These lipid modifications have often been implicated in membrane association of the modified proteins (5). Palmitoylation occurs posttranslationally. Moreover, rapid turnover of the protein-bound fatty acid has been shown for several proteins (6 -8). The recent surge of interest in studying protein palmitoylation is largely due to the fact that a number of proteins involved in intracellular signaling are palmitoylated, and this appears to be regulated by the activation status of these proteins (9 -12). Palmitoylation occurs in, among others, the families of (i) G protein-linked receptors, (ii) the subunits of heterotrimeric G proteins, and (iii) the non-receptor tyrosine kinases. Palmitate plays a role in the membrane association of these polypeptides.Moreover, recent evidence suggests that this may regulate protein-protein interactions such as those between growth cone-associated protein 43 and G o␣ in neurons (13). Although evidence that the interactions of G proteins with effectors are regulated by palmitoylation is becoming compelling, very little is known about the enzymes involved in the catalysis of the palmitoylation and depalmitoylation of these or other proteins.A clear understanding of the palmitoylation-depalmitoylation cycle as a cellular control mechanism obviously entails characterization of the protein palmitoyltransferase(s) and thioesterase(s). A thioesterase has recently b...

Protein myristoylation in health and disease

“…It has been shown that myristoyl peptides bind membranes with a Gibbs free binding energy of around 8 kcal/mol [73], whereas the more hydrophobic palmitoylCoA binds more strongly, resulting in around 15-fold greater partitioning of palmitoylated substrates to the membrane [72]. Palmitoyl acyltransferases have only recently been characterised, but in most cases studied they also localise to membranes [74,75]. This could be one mechanism by which certain fatty acyl-CoA substrates are made unavailable to NMT in the cell.…”

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase

“…Despite the accumulation of evidence showing that the palmitoylation of various proteins, including RAS, is critical for their biologic functions, the enzymes involved in this process are still poorly understood. Several groups have sought to purify PAT enzymes (Gutierrez and Magee, 1991;Liu et al, 1996;Das et al, 1997;Ueno and Suzuki, 1997;Hiol et al, 2003), unfortunately none have provided any examples of bona fide human Pat enzymes.…”

Palmitoylation of Caveolin-1 in Endothelial Cells Is Post-translational but Irreversible

“…The reversibility of protein-palmitate bonds has been demonstrated by the rapid turnover of the palmitoyl group and may be caused by the relative lability of the thioester linkage (8 -10). Protein palmitoylation and depalmitoylation reactions are considered to be primarily enzymatic and catalyzed by unidentified membrane-bound palmitoyl acyltransferases (11,12) and by cytoplasmic and lysosomal forms of palmitoyl thioesterases (13)(14)(15), respectively. Regulation of the protein palmitoylation and depalmitoylation cycle is thought to be important in regulating protein localization, conformation, protein-protein interaction, and activity (16 -18).…”