Summary Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here, a global characterization of the neuronal palmitoyl-proteome identifies most of the known neuronal palmitoyl-proteins (PPs), 68 in total, plus over 200 new PP candidates, with additional testing confirming palmitoylation for 21 of these candidates. New PPs include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is a finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl) both with regard to localization and function: Cdc42-palm, concentrates in dendritic spines and plays a special role in inducing these post-synaptic structures. Finally, assessing palmitoylation dynamics in drug-induced activity paradigms finds rapidly induced changes both for Cdc42 as well as for other synaptic PPs, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

As a reversible posttranslational modification, protein palmitoylation has the potential to regulate the trafficking and function of a variety of proteins. However, the extent, function, and dynamic nature of palmitoylation are poorly resolved because of limitations in assay methods. Here, we introduce methods where hydroxylamine-mediated cleavage of the palmitoyl-thioester bond generates a free sulfhydryl, which can then be specifically labeled with sulfhydryl-reactive reagents. This methodology is more sensitive and allows for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or nonradioactive, and can be used to assay the palmitoylation of proteins expressed in vivo in brain or other tissues.

Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.

Nicotinic acetylcholine receptors in the nervous system are heterogeneous with distinct pharmacological and functional properties resulting from differences in post-translational processing and subunit composition. Because of nicotinic receptor diversity, receptor purification and biochemical characterization have been difficult, and the precise subunit composition of each receptor subtype is poorly characterized. Evidence is presented that alpha-bungarotoxin (Bgt)-binding nicotinic receptors found in pheochromocytoma 12 (PC12) cells are pentamers composed solely of alpha7 subunits. Metabolically labeled, affinity-purified Bgt receptors (BgtRs) consisted of a single 55 kDa band on SDS gels, which was recognized by anti-alpha7 antibodies on immunoblots. Isoelectric focusing separated the 55 kDa band into multiple spots, all recognized by anti-alpha7 antibodies and, therefore, each a differentially processed alpha7 subunit. Cell-surface BgtR subunits, cross-linked to each other and (125)I-Bgt, migrated on gels as a ladder of five bands with each band a multiple of an alpha7 subunit monomer. Similar characteristics of BgtRs from rat brain suggest that they, like PC12 BgtRs, are alpha7 pentamers containing differentially processed alpha7 subunits.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, due to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated α-isoform to the postsynaptic density (PSD) and the L27 domain-containing β-isoform primarily to non-PSD, perisynaptic regions. Consequently, α- and βSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs, and hence their responsiveness to presynaptically released glutamate.

We have characterized the ␣-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of ␣7-containing receptors that bind ␣-Bgt with high affinity (K d ϭ 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and ␣-Bgt block ϳ90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric ␣7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for ␣7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and ␣7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and ␣7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

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