[Show abstract][Hide abstract] ABSTRACT: Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.
[Show abstract][Hide abstract] ABSTRACT: The addition of a lipid moiety to a protein increases its hydrophobicity and subsequently its attraction to lipophilic environments like membranes. Indeed most lipid-modified proteins are localized to membranes where they associate with multiprotein signaling complexes. Acylation and prenylation are the two common categories of lipidation. The enzymology and pharmacology of prenylation are well understood but relatively very little is known about palmitoylation, the most common form of acylation. One distinguishing characteristic of palmitoylation is that it is a dynamic modification. To understand more about how palmitoylation is regulated, we fused palmitoylation substrates to fluorescent proteins and reported their subcellular distribution and trafficking. We used automated high-throughput fluorescence microscopy and a specialized computer algorithm to image and measure the fraction of palmitoylation reporter on the plasma membrane versus the cytoplasm. Using this system we determined the residence half-life of palmitate on the dipalmitoyl substrate peptide from GAP43 as well as the EC(50) for 2-bromopalmitate, a common inhibitor of palmitoylation.
Methods in Enzymology 02/2006; 414:150-87. · 2.00 Impact Factor