Characterization and partial p

acyltransferase activ arle ive an onnec ylvani form A wide variety of cellular and viral proteins undergo thioester bond. The dynamic nature of palmitoylation makes Biochimica et Biophysica Acta 16properties of a protein by changing its protein interactions, affinity and orientation toward the membrane and targeting within membranes. The two most common forms of acyl-ation are myristoylation that occurs on amino-terminal In contrast to myristoylation, in which the N-myristoyl transferase is well-characterized, the enzymology of palmi-toylation is less clear. A strong candidate for the enzyme that removes palmitate is acyl protein thioesterase 1 [5,6], a member of the a/h hydrolase family [7]. Purification of a protein fatty acyltransferase (PAT) has proven much more difficult because: (1) the enzymatic activity is labile and lost early in the purification process [8,9]; (2) in vitro PAT activity has been shown for proteins that are or are likely Abbreviations: CoA, coenzyme A; cpm, counts per minute; PAT, protein fatty acyltransferase; RGS16, regulator of G protein signaling 16; S.E., standard error of the mean; SDS-PAGE, sodium dodecyl sulfate-fatty acids (reviewed in Refs. [1–4]). Acylation can alter theposttranslational modification by the covalent attachment of it a critical regulator of G protein signaling, synaptic transmission and other cellular processes.plasma membrane fraction to minimize the contamination with these enzymes. After detergent extraction of the plasma membrane fraction, the PAT activity was enriched about 90-fold by sequential chromatography including affinity chromatography to a cerulenin-based inhibitor of palmitoylation. The partially purified PAT activity (1) was lost with treatments to degrade or denature proteins, (2) could acylate tubulin, Gai and RGS16 and (3) showed a preference for palmitate and to a lesser degree other long-chain fatty acids. This purification procedure is a significant advance over previous efforts at PAT purification and a starting point for a proteomic approach for identification of mammalian PAT