Wild-type huntingtin plays a role in brain development and neuronal survival. and enhanced neuronal toxicity and cell Bergaptol death (9). In addition to that of HTT, palmitoylation and function of glial glutamate transporter-1 (GLT-1) are also reduced in the YAC128 mouse model of HD. Impaired GLT-1 palmitoylation is present early in the pathogenesis of HD and may contribute to decreased glutamate uptake, excitotoxicity and, ultimately, neuronal cell death in HD (10). Multiple studies in cellular and animal model systems indicate that mutant HTT imparts a novel toxic function, and the mechanism of toxicity involves multiple pathogenic pathways (11). However, the normal function of FLJ44612 wild-type HTT, an essential cellular protein in higher vertebrates, is not yet well understood (12). HTT is encoded by a single gene and is ubiquitously expressed in mammals. It is primarily a cytoplasmic protein, known to be both vesicle- and microtubule-associated (13,14). Deletion of the gene is lethal early in mouse embryogenesis (15C17). The multi-domain structure of HTT indicates that the protein may have multiple distinct cellular roles, including transcriptional regulation, Bergaptol nucleo-cytoplasmic shuttling, vesicular trafficking and intracellular transport in the cell, synaptic function and anti-apoptotic activity (reviewed in 12,18). Accumulating data on the roles of wild-type HTT in endocytosis, endosomal motility and axonal transport have led to an emerging model for HTT as an integrator of protein transport along the cellular cytoskeleton (reviewed in 12). HIP14, which is predominantly expressed in the Golgi apparatus, is also found to be associated with endosomal vesicles in the soma, presynaptic terminals and dendritic spines. It is possible that trafficking of these HIP14-containing Bergaptol vesicles to sites where it executes its enzymatic function may require HTT. Given the multi-domain nature of HTT and its strong interaction with HIP14, HTT may also function as an allosteric activator of HIP14. In this study, we tested the hypothesis that wild-type HTT, in addition to serving as a substrate of HIP14, also directly influences HIP14 enzymatic activity. RESULTS HIP14 and HIP14L are the two major HTT PATs We have previously shown that HTT is palmitoylated by HIP14 and HIP14L (DHHC-13), a HIP14 homolog (3,8,9). In fact, palmitoylation of HTT by HIP14 is essential for its trafficking and function (9). The identification of 23 DHHC proteins in this PAT family raised the question whether other DHHC proteins palmitoylate HTT (5). We therefore applied the acyl biotin exchange (ABE) labeling method to examine the palmitoylation level of HTT upon overexpression of individual DHHC proteins along with HTT in COS cells. We found that HIP14 and HIP14L are the two major PATs that significantly increase palmitoylation of HTT (Fig.?1A?and B). Open in a separate window Figure?1. HIP14 and HIP14L are the two major PATs for HTT. (A) COS cells expressing either HTT alone Bergaptol or HTT with individual DHHC protein. HTT was immunoprecipitated, followed by the biotin palmitoylation labeling assay. Among the indicated DHHC proteins, HIP14L (DHHC-13) and HIP14 (DHHC-17) significantly increased palmitoylation of HTT. (B) Quantification of HTT palmitoylation increase by individual DHHC protein. Palmitoylation of HTT by an individual enzyme was normalized by western blot and compared with control. Number of repeats Bergaptol was indicated in the graph. (C) Interaction between PSD95 and HIP14 requires the presence of palmitoylated cysteines C3, 5. Mutation of cysteines 3 and 5 in PSD95 abolished its interaction with HIP14 detected by co-IP. (D) In contrast, association of wild-type HTT to HIP14 was independent of palmitoylation of HTT. Interaction of HIP14 and HTT is unique and distinct from other palmitoylation enzymeCsubstrate interactions Co-transfection of a palmitoylation substrate protein with its cognate PAT has been found in a number of cases to result.