Protein Palmitoylation by DHHC Protein Family

Palmitoylation is the post-translational modification of proteins with palmitic acid (16-carbon saturated fatty acid) and regulates the membrane targeting, subcellular trafficking and function of proteins [1]. Palmitoylation occurs either through amide-linkage (N-palmitoylation) or thioester linkages (S-palmitoylation). S-palmitoylation occurs on cysteine residues in diverse sequence contexts and is more commonly found in most palmitoylated proteins. Here, the term of protein palmitoylation will mean S-palmitoylation. Protein palmitoylation is the frequent lipid modification of neuronal proteins and modifies many important proteins, including synaptic vesicle proteins, ion channels, guanosine triposphate (GTP)-binding proteins, neurotransmitter receptors and synaptic scaffolding proteins [2]. Examples include PSD-95, a protein that scaffolds receptors and signaling enzymes at the post-synapse; NCAM140, a neural cell adhesion molecule that localizes at the growth cone and regulates neurite outgrowth; and SNAP-25, a t-SNARE protein that regulates neurotransmitter release [2]. PSD-95 palmitoylation is necessary for sorting of PSD-95 to dendrites and participates in the post-synaptic clustering of PSD-95 in dendritic spines, thereby regulating the clustering of α-amino-3-hydroxy-5-methyl-4-isox-azole propionic acid (AMPA)-type glutamate receptors [3]. Unlike other irreversible lipid modifications such as myristoylation and prenylation, palmitoylation is relatively labile and palmitate on proteins turns over rapidly. Importantly, the specific extracellular signal regulates protein-palmitoylation levels [4]. At post-synaptic sites, palmitate continuously turns over on PSD-95. Depalmitoylation of PSD-95 is enhanced by glutamate receptor-mediated synaptic activity, and this process dissociates PSD-95 and AMPA receptors from the postsynaptic sites [3]. Although the actions of enzymes that add or remove protein palmitate might mediate the dynamic regulation of palmitoylation, the enzymes have been elusive. Recent genetic studies in yeast have identified proteins that mediate palmitoylation. Erf2p [5–7] and Akr1p [8] are palmitoyl-transferases (PATs) for yeast Ras2p and yeast casein kinase2 (Yck2p), respectively (Figure 5.1). Erf2p and Akr1p are integral membrane proteins harboring a cysteine-rich domain containing a conserved DHHC (Asp-His-His-Cys) motif. In genomes of human and mouse, 23 kinds of DHHC-containing proteins are predicted (Figure 5.2). To identify the physiological PATs for substrates, systemically evaluating functions of the family of 23 DHHC-containing proteins is necessary. For this purpose, we isolated all mouse DHHC proteins and established a screening method that allows us to identify the candidate PAT for specific substrates [9]. We found that a subset of DHHC proteins specifically palmitoylates PSD-95 (P-PATs), and that DHHC proteins have substrate specificity (Figure 5.2) [9]. P-PAT activity regulates synaptic clustering of PSD-95 and AMPA receptors, as well as modulating AMPA receptor function in hippocampal neurons. Thus, the DHHC protein-mediated reaction is a potential general mechanism of protein palmitoylation in cells. In this chapter, we describe procedures to screen the DHHC protein family to identify the specific PAT. The procedures include three steps.