Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells

Abstract

Posttranslational modifications, including phosphorylation, ubiquitination and lipid modifications, provide proteins with additional functions and regulation beyond genomic information. Palmitoylation is a reversible lipid modification with palmitic acid that plays critical roles in protein trafficking and function. However, the enzymes that mediate palmitoyl acyl transferase (PAT) have been elusive. Recent genetic analysis in yeast revealed that members of cysteine-rich DHHC domain containing proteins (DHHC proteins) mediate palmitoylation. In mammalian genomes, 23 DHHC proteins are predicted raising the possibility of a large family of PAT enzymes. Here, we describe a systematic method to examine which of the DHHC family members is responsible for palmitoylation of a substrate.

Introduction

Protein palmitoylation, a lipid modification, plays a pivotal role in protein trafficking and function [1], [2]. 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 found more commonly in most of palmitoylated proteins. Here, the term of protein palmitoylation will mean S-palmitoylation. Substrates for palmitoylation include many important proteins; GTP-binding proteins, cytoskeletal proteins, enzymes, neurotransmitter receptors, and synaptic scaffolding proteins. Examples include; H-Ras, a small GTP-binding protein that regulates cell growth and cell differentiation [3], SNAP-25, a t-SNARE protein that regulates neurotransmitter release [4] and PSD-95, a protein that scaffolds receptors and signaling enzymes at the postsynapse (Fig. 1) [5]. Unlike other irreversible lipid modifications such as myristoylation and prenylation, palmitoylation is relatively labile and palmitate on proteins turns over rapidly. Importantly, extracellular signals regulate protein palmitoylation levels, possibly through the activity of PATs and/or palmitoylthioesterases [6], [7]. At postsynaptic 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 type glutamate receptors from postsynaptic sites [7]. Thus, the reversible nature of protein palmitoylation enables cells to exert their dynamic function in response to extracellular signals.

Despite the huge importance of protein palmitoylation, the enzymes that add palmitate to proteins and cleave the thioester bond have been elusive for a long time. Recent elegant forward genetic approaches in yeast have shed light on this issue. Erf2p [8], [9], [10] and Akr1p [11] were identified as PATs for yeast Ras2p and yeast casein kinase2 (Yck2p), respectively. Interestingly, Erf2p and Akr1p have common topology and motif, sharing several transmembrane domains and an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) (here, referred to as a DHHC domain) [2], [12]. The DHHC domain represents a palmitoyl transferase domain as mutations in this domain abolish PAT activity. In human and mouse genomes, 23 DHHC proteins are predicted (Fig. 2). Although the sequence similarity is highest in the DHHC domain, each member has several (at least four) transmembrane domains (Fig. 3). To identify the physiological PATs for particular substrates, it is necessary to systematically evaluate the functions of all 23 DHHC proteins. For this purpose, we isolated all of the mouse DHHC proteins and established a screening method that allows us to identify candidate PATs for particular substrates [13]. We found that a subset of DHHC proteins (P-PATs) specifically palmitoylate PSD-95 and that DHHC proteins have substrate specificity (Fig. 2, Fig. 4). 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 mammalian cells, as well as in yeast. Here, we describe a simple, systematic and straightforward method to identify candidate PAT:substrate pairs. The procedure includes three steps: (1) transfection of the target substrate cDNA together with individual DHHC clone in a heterologous culture system, (2) metabolic labeling with [³H]palmitic acid, (3) SDS–PAGE and fluorography. Most of the techniques described here were originally used for identification of P-PATs. This procedure has enabled us to further identify the PATs for over 10 different substrates.