Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Roxanne A. Vaughan,James D. Foster

doi:10.1074/jbc.m110.187872

Abstract

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

Highlights

Identification of dopamine transporters (DATs) Palmitoylation—LLCPK1 cells stably transfected with wild type (WT) rDAT were metabolically labeled with 10 ␮M [3H]palmitic acid for 1–18 h, and DATs were extracted by immunoprecipitation and subjected to SDS-PAGE/fluorography
We demonstrate that DAT undergoes thioacylation with palmitic acid and show that this modification exerts multiple layers of control over the protein via impacts on transport capacity, degradation, and protein kinase C (PKC)-dependent regulation
With respect to longer term regulation of DAT by palmitoylation, we found that higher dose/ longer duration 2BP treatments in both synaptosomes and cells caused DAT protein losses and that these treatments and C580A mutation resulted in production of DAT degradation fragments, supporting an additional role for palmitoylation in opposing DAT turnover

Summary

Introduction

Rat striatal synaptosomes were treated with vehicle (no treatment) or the indicated concentrations of palmitate (Pal) or 2BP at 30 °C for 60 min, and aliquots were analyzed for [3H]DA transport, [3H]alanine transport, or DAT immunoblotting. B, equal amounts of protein from treated synaptosomes (conditions indicated directly below on histogram) were immunoblotted for DAT (representative blot shown), and band density was expressed as a fraction of control values normalized to 100% (means Ϯ S.E. of three experiments performed in duplicate).

Results

Conclusion