Palmitoylation of ATP-Binding Cassette Transporter A1 Is Essential for Its Trafficking and Function
ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.
The ATP-binding cassette transporter (ABC)A1 is essential for the transport of lipids across membranes and for the subsequent formation of high-density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, which is characterized by low to absent plasma HDL cholesterol and deposition of lipids in the reticuloendothelial cells of those affected.1–3
ABCA1 localizes to both the plasma membrane and to intracellular compartments within a cell.4 The plasma membrane and endosomes are the 2 most important reservoirs of cellular cholesterol.5 Approximately 20% of newly synthesized apolipoprotein (Apo)A-1 from Hep G2 cells is lipidated before secretion highlighting the important role of intracellular lipidation.6 Cholesterol deposits in late endosomes and lysosomes are important sources of lipids in ABCA1-mediated cholesterol efflux.7
Several naturally occurring mutations of ABCA1 that result in its mislocalization from the plasma membrane lead to lipid efflux deficiency.8 The presence of high-affinity binding sites for ApoA-1 on the cell surface,9 as well as the ability of lipid-signaling molecules such as ceramide to increase ABCA1 expression at the plasma membrane, resulting in increased cholesterol efflux,10 also highlights the importance of cell surface ABCA1 for its activity.
ABCA1 rapidly recycles between the cell surface and intracellular compartments.11,12 ABCA1 trafficking to specific intracellular and plasma membrane sites is likely to be exquisitely controlled, because this localization is essential for its function and vital for the normal regulation of intracellular cholesterol. However, very little is known about the processes regulating specific subcellular localization of ABCA1.
An amino-terminal signal anchor sequence functions to properly orient ABCA1 in the endoplasmic reticulum membrane following translation.13 The purported role that this signal sequence plays in targeting of ABCA1 to the cell surface or various organelles is ill-defined. ABCA1 contains a cytoplasmic domain PEST sequence that is important in ABCA1 internalization and trafficking to late endosomes and lysosomes. The deletion of this PEST sequence retains ABCA1 at the plasma membrane and results in impaired cholesterol efflux from late endosomal cholesterol pools.12
Palmitoylation, which is the posttranslational modification of proteins with the lipid palmitate, has recently emerged as an important modification that regulates protein trafficking and function by targeting proteins to various cellular compartments or specific domains in the plasma membrane.14 Palmitate is a 16-carbon saturated fatty acid that attaches to proteins posttranslationally. This modification increases the hydrophobicity of a protein, facilitates protein interactions with lipid bilayers, and can markedly alter protein sorting and function. Whereas other lipid modifications, such as myristoylation or isoprenylation, are permanent, the thioester bond that links proteins to palmitate is labile and reversible15–17 and thus is a process that has the potential to exquisitely regulate protein function.
Here, we show ABCA1 to be robustly palmitoylated and that lack of ABCA1 palmitoylation affects its normal distribution and its cholesterol and phospholipid efflux capabilities. Multiple enzymes including most prominently DHHC8 are involved in the palmitoylation of ABCA1 and on increasing DHHC8 expression, an increase in ABCA1-mediated cholesterol efflux activity is observed. This study involving ABCA1 palmitoylation represents a novel target for increasing ABCA1 activity independent of transcriptional mechanisms.
Materials and Methods
Generation of ABCA1 Constructs
pcDNA3-ABCA1 was generated by RT-PCR amplification of human ABCA1 from liver RNA. ABCA1 was sequence verified and enhanced green fluorescent protein (EGFP) (Clontech) was added as a C-terminal fusion. This fusion did not cause mislocalization of ABCA1 nor alter its lipid efflux capabilities.8 All palmitoylation mutations in ABCA1 were generated by PCR-based site-directed mutagenesis as previously described18 in the pcDNA-ABCA1-EGFP vector and were fully sequenced. They were then cloned into the pcDNA5-FRT vector (Invitrogen) to generate stable cell lines. N-terminal ABCA1 constructs were generated by restriction enzyme digests and subcloning into the pcDNA3.1 vector.
Construction of Stable Cell Lines Expressing ABCA1 Variants
Polyclonal ABCA1 stable cell lines were generated in 293 Flp-In cells (Invitrogen) by cotransfecting pcDNA5/FRT-ABCA1 and pOG44 (Invitrogen) according to the recommendations of the manufacturer. Hygromycin-resistant colonies were evaluated for ABCA1 expression by Western blot analysis. A control hygromycin-resistant cell line was generated with the empty pcDNA5/FRT vector.8
Protein Isolation and Antibodies
Cells were lysed in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EGTA, 1 mmol/L EDTA, 1% Triton X-100, 0.2% sodium dodecyl sulfate [SDS]). A total of 40 to 60 μg of protein was separated on 7.5% acrylamide gels and transferred onto poly(vinylidene difluoride) (PVDF) membranes (Millipore). ABCA1 was immunodetected using an anti-ABCA1 monoclonal antibody generated to its C terminus.19 In addition, anti-myc (in-house), anti-His (Roche), anti-GFP (gift from Luc Berthiaume), anti-phalloidin (Invitrogen), anti-GAPDH (Chemicon) primary antibodies, anti-mouse and anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Bio-Rad), and anti-mouse Alexa Fluor 488 (Molecular Probes) secondary antibody were used.
2-Bromopalmitate Treatment of Cells to Inhibit Protein Palmitoylation
A 100 mmol/L stock of 2-bromopalmitate (2BP) (Sigma) was prepared in DMSO (Sigma) according to the instructions of the manufacturer. The stock solution was diluted into media to a final concentration of 60 μmol/L. Cells were incubated from 5 to 8 hours with 2BP before preparation for immunofluorescence imaging or cholesterol efflux assays.
Transiently transfected COS, HEK 293, or stable ABCA1 Flp-In cells were labeled with 1 to 2.5 mCi/mL [3H]palmitic acid (57 Ci/mmol, Perkin Elmer) for 3 hours and processed as previously described.20 For immunoprecipitation of full-length ABCA1, anti-ABCA1 AC10 monoclonal antibodies were used. N-terminal ABCA1 was immunoprecipitated with anti-His antibody. Protein G Sepharose beads were used for immunoprecipitation. Alternately, a method using acyl-biotinyl exchange chemistry21 was used where immunoprecipitated ABCA1 was subjected to 3 steps of acyl-biotinyl exchange chemistry: (1) N-ethylmaleimide to block free protein thiols; (2) release of palmitoyl modification through hydroxylamine-mediated cleavage of the thioester linkage; (3) biotinylation of exposed palmitoylation site thiols with biotin-conjugated 1-biotinamido-4-[4`-(maleimidomethyl)cyclohexanecarboxamide] butane (Biotin-BMCC). Finally, ABCA1 protein was eluted and subjected to Western analysis using streptavidin–horseradish peroxidase. In addition, metabolic labeling with 125I-iodopalmitate was used. Synthesis of the iodopalmitate analog was carried out as previously described.22 Briefly, the radioiodination of 16-iodo-hexadecanoic acid was carried out by incubation with Na125I-iodide to generate the palmitate analog 16-125I-iodohexadecanoic acid (125I-iodopalmitate). HEK 293 stable cell lines expressing ABCA1 were metabolically labeled with 125I-iodopalmitate (25 to 50 μCi/mL) for 4 hours. Samples were separated on 8% SDS-PAGE separating gels and transferred onto PVDF (Millipore) membranes. 125I-iodopalmitate incorporation was visualized by phosphorimaging and autoradiography. PVDF membranes were hydrolyzed by soaking in 0.2N KOH (pH ≈13.0) for 24 hours and reexamined by phosphorimaging and autoradiography. Membranes were subsequently processed for immunoblotting with anti-ABCA1 antibody.
Following metabolic labeling of cells expressing ABCA1 with [3H]palmitic acid, cell lysates were immunoprecipitated and treated with either 1 mol/L Tris-HCl (pH 7.5) or 1 mol/L NH2OH (hydroxylamine) for 1 to 2 hours at 25°C.
Cell Surface Biotinylation
Transiently transfected COS cells were surface labeled with EZ-Link Sulfo-NHS-SS-Biotin (Pierce) for 30 minutes at 4°C. Cells were then lysed in lysis buffer (10 mmol/L Tris [pH 8.0], 1% Triton X-100). Immobilized NeutrAvidin gel (Pierce) was added to the lysate. The gel was washed with lysis buffer and samples were separated on 8% SDS-PAGE gels and transferred onto PVDF membranes (Millipore). Membranes were immunoblotted with anti-ABCA1 and anti-GAPDH monoclonal antibodies. GAPDH was run as a control to ensure internalization of the biotin label did not occur. Images were quantified using Quantity One (Bio-Rad).
Lipid Efflux Essay
ABCA1 Flp-In cells were plated onto 24-well dishes in DMEM/10% FBS/50 U/mL penicillin-streptomycin/20 mmol/L l-glutamine. Twenty-four hours later, 10 μCi/mL choline chloride or 1 μCi/mL cholesterol (Perkin-Elmer) was added. After 16 hours, cells were equilibrated in DMEM/0.2% defatted BSA, followed by efflux to 10 to 20 μg/mL apolipoprotein (Apo)A-I (Lee Biosystems). Lipids were extracted from the supernatants and cell lysates using chloroform:methanol, and the radioactivity was quantified. Efflux is expressed as a percentage of the efflux induced by wild-type (WT) ABCA1.
Transiently transfected COS cells were fixed in 2% paraformaldehyde, 2% sucrose in PBS and permeabilized in 0.3% Triton X-100 (Sigma). Cells were stained with GFP antibody, followed by incubation with a secondary antibody conjugated to Alexa 488 fluorophore and by incubation with phalloidin conjugated to Alexa 568 fluorophore. Cells were mounted with fluorescent mounting medium (DakoCytomation). Images were acquired on a Carl Zeiss Laser Scanning System LSM 510 META confocal microscope and handled using the Zeiss LSM Image Browser.
All statistical analyses were performed using the 2-tailed Student’s t test using GraphPad Prism (GraphPad Software). Results are plotted as percentage±SD.
Acute Inhibition of Palmitoylation Disrupts Normal ABCA1 Trafficking and Lipid Efflux
Because localization of ABCA1 at the plasma membrane is vital for its lipid transport function, we hypothesized that palmitoylation may play a role in the targeting of ABCA1 to the plasma membrane. To determine whether palmitoylation regulates ABCA1 transport and function, we treated ABCA1-overexpressing cells with 2-BP, a nonhydrolyzable analog of palmitate that renders palmitoylation nonfunctional.23 In the presence of 2-BP, ABCA1 did not localize to the plasma membrane in COS and hepG2 cells (Figure 1A and 1B), indicating that palmitoylation may contribute to the proper subcellular localization of ABCA1.
Because plasma membrane localization of ABCA1 was inhibited by the palmitoylation blocker 2-BP, we next assessed whether ABCA1-mediated cholesterol efflux to ApoA-I was affected by the acute inhibition of palmitoylation. Indeed, in the presence of 2-BP, ABCA1-mediated cholesterol efflux to ApoA-I was significantly reduced, indicating that abrogation of palmitoylation results in reduced ABCA1 efflux activity (Figure 1C).
ABCA1 is Palmitoylated
Although the above studies using 2-BP suggested that palmitoylation of ABCA1 is important for its localization and efflux function, this treatment results in the acute inhibition of palmitoylation of many substrates. Thus, the above experiments did not directly or specifically address the role of palmitoylation of ABCA1. To directly determine whether ABCA1 is palmitoylated, ABCA1 was immunoprecipitated from cells, and palmitoylation was assessed using biotinylated BMCC. In the presence of hydroxylamine, a reagent that specifically cleaves palmitate, a robust signal was observed, indicating that ABCA1 is palmitoylated (Figure 2A).
To confirm ABCA1 palmitoylation, 2 additional methods were used: I125-palmitate incorporation (Figure 2B) or [3H]palmitate incorporation (Figure 2C) by metabolic labeling. Using these additional methods, ABCA1 is robustly palmitoylated, confirming our previous finding. Treatment with KOH (Figure 2B, right) or hydroxylamine (Figure 2D) resulted in significant reductions in palmitoylation signal, indicating that the binding of palmitate to ABCA1 was specific, because both KOH and hydroxylamine hydrolyze the palmitate thioester bond.24
ABCA1 Is Palmitoylated in Its N Terminus
Palmitoylation occurs on intracellular cysteine residues, usually close to transmembrane domains.25 However, no consensus sequences for palmitoylation are currently known. ABCA1 contains 39 cysteines, of which 23 occur in predicted intracellular regions. To determine the site of palmitoylation of ABCA1, we generated an N-terminal construct of ABCA1 that consisted of amino acids 1 to 639. Using [3H]palmitate metabolic labeling, the N terminus of ABCA1 was robustly palmitoylated (Figure 2E). Thus, ABCA1 is palmitoylated in its N terminus.
ABCA1 Is Palmitoylated at Cysteine Residues 3 and 23
The N-terminal fragment of ABCA1 that showed robust palmitoylation contains 11 cysteines. Of these, 2 cysteines are in the intracellular region, and 9 are localized to the large extracellular loop (Figure 3A and 3B). Because palmitoylation occurs in intracellular regions, we replaced by site-directed mutagenesis the 2 cysteines at amino acids 3 and 23 with serines. Three mutant fragments were generated: C3S, C23S, and the double C3/C23S. In vitro metabolic labeling assays using [3H]palmitate showed that palmitoylation occurs on both the C3 and C23 residues, because ABCA1 palmitoylation levels were reduced in all 3 mutant constructs (Figure 3C). However, compared with the mutation at C23, mutation at C3 resulted in only a minor reduction in N-terminal ABCA1 palmitoylation, indicating that of the 2 cysteines, C23, the cysteine closest to the transmembrane domain, is the major site of palmitoylation. Thus, ABCA1 is palmitoylated at amino acids 3 and 23.
ABCA1 Is Also Palmitoylated at Cysteines 1110 and 1111
Although palmitoylation occurs on cysteines 3 and 23 in the N terminus of ABCA1, the possibility of palmitoylation in the rest of ABCA1 (amino acids 640 to 2261) had not been assessed. To determine whether cysteines 3 and 23 are the only sites of palmitoylation in ABCA1, we generated these mutations in full-length ABCA1. Using [3H]palmitate metabolic labeling, we found that in the presence of the C3 and C23 mutations, full-length ABCA1 still showed robust palmitoylation (Figure 3D), indicating the presence of additional palmitoylation sites.
Because palmitoylation occurs in intracellular cysteines often within close proximity to transmembrane domains, and several proteins are palmitoylated at double cysteine sites,25 we first generated cysteine to serine mutants of the adjacent amino acids 1110 and 1111 of ABCA1. Assessment of palmitoylation of these sites in the context of full-length ABCA1 showed that ABCA1 was still palmitoylated (Figure 3D, lane C1110/C1111S). However, generation of the quadruple mutant C3/23/1110/1111S in full-length ABCA1 resulted in a significant reduction in ABCA1 palmitoylation (Figure 3D, lane C3/C23/C1110/C1111S; and Figure 3E). Thus, cysteines at residues 3, 23, 1110, and 1111 are major sites of palmitoylation in ABCA1.
Palmitoylation of ABCA1 at C3, -23, -1110, and -1111 Is Essential for Its Proper Trafficking
ABCA1 is localized in the endoplasmic reticulum, in endocytic vesicles, and at the plasma membrane. Because palmitoylation ensures the proper sorting and targeting of proteins, we assessed whether the palmitoylation defective ABCA1 showed altered trafficking. Indeed, each of the palmitoylation-deficient mutants resulted in ABCA1 that did not reach the plasma membrane and, instead, was localized in intracellular regions (Figure 4). Mutation of all palmitoylation sites combined, C3, -23, -1110, and -1111 also resulted in a similar subcellular localization, with ABCA1 not present at the plasma membrane. As a control, cells expressing the ABCA1 mutant M1091T were used. This mutation has been previously described in patients and represents a severe loss-of-function mutation, resulting in a marked decrease in ABCA1 expression at the plasma membrane.8 In addition, this mutation is thought to act in a dominant negative manner because patients heterozygous for this mutation have a homozygote-like phenotype.8
Palmitoylation of ABCA1 Regulates Its Localization at the Plasma Membrane
In addition to the assessment of ABCA1 localization by immunofluorescence, we performed cell surface biotinylation on WT and C3/23/1110/1111S to quantify the levels of plasma membrane ABCA1. In agreement with our immunofluorescence data, the cell surface expression of palmitoylation-deficient ABCA1 was reduced by >90% compared to WT ABCA1 (Figure 5A and 5B), indicating that the palmitoylation of ABCA1 is essential for its plasma membrane localization.
ABCA1 Palmitoylation Is Essential for Cholesterol and Phospholipid Efflux
We next assessed whether the palmitoylation of ABCA1 was essential for its function in lipid transport. We generated stable cell lines harboring each of the C3S, C23S, C1110S, and C1111S mutants singly and in combination and determined the extent of both cholesterol and phospholipid efflux to ApoA-I facilitated by these cell lines. In the presence of any of the cysteine mutations, ABCA1-mediated cholesterol (Figure 5C) and phospholipid (Figure 5D) efflux to ApoA-I was significantly decreased. However, the abrogation of palmitoylation at each of these sites resulted in a similar magnitude (40% to 60%) of decrease in efflux, as did the abrogation of palmitoylation at all 4 cysteines. This result is not surprising, considering our finding that abrogation of palmitoylation at each of the palmitoylation sites alone results in the mislocalization of ABCA1 away from the plasma membrane and its accumulation in intracellular sites. In addition, our data indicate that palmitoylation at these sites, although important, is not the only factor regulating ABCA1 trafficking. As a control, efflux from a stable cell line harboring the naturally occurring ABCA1 mutation M1091T that was previously characterized as severe loss-of-function mutation8 was reduced by ≈80%.
DHHC8 Is a Major Palmitoyl Transferase for ABCA1
In mammals, palmitoylation is catalyzed by a family of 23 DHHC domain containing palmitoyl transferases (PATs).26,27 To determine the palmitoyl transferase for ABCA1, we obtained and transiently transfected each of the PATs into an ABCA1 overexpressing cell line. Using [3H]palmitate metabolic labeling, we found that DHHC8 most significantly increased the palmitoylation of ABCA1 (Figure 6A). DHHC2, -12, -15, -20, and -21 were also able to increase ABCA1 palmitoylation, although these increases were modest (Figure 6A).
Treatment of palmitoylated ABCA1 with hydroxylamine removed much of the palmitate signal, indicating that the increase in palmitoylation caused by overexpression of the PATs was specific and not caused by nonspecific association of palmitate with ABCA1 protein (Figure 6B).
ABCA1-Mediated Efflux Is Increased in the Presence of Its PAT, DHHC8
Because DHHC8 is the major PAT for ABCA1, we analyzed its ability to increase ABCA1-mediated efflux. When DHHC8 was transiently transfected into an ABCA1-expressing stable cell line, cholesterol efflux to ApoA-I was significantly increased (Figure 7A). Transient transfection of DHHC2 and -12, 2 other PATs that had mild effects on ABCA1 palmitoylation, did not result in increased ABCA1-mediated efflux, indicating that these were not the major PATs for ABCA1. Thus, increasing DHHC8 mediated palmitoylation of ABCA1 results in increased ABCA1 function.
Because it is possible that one other cause for the observed increase in cholesterol efflux may be an increase in ABCA1 expression modulated by the overexpression of PATs, we assessed ABCA1 protein in the presence of PATs DHHC2, -8, and -12. ABCA1 protein levels were not altered in the presence of any of the 3 PATs (Figure 7B). Thus, the increase in efflux mediated by DHHC8 was not attributable to increased ABCA1 protein but more likely caused by increased ABCA1 palmitoylation and increased presence of ABCA1 at its subcellular sites of function.
PATs are widely thought to interact with their substrates. We next performed immunoprecipitation experiments with ABCA1 and DHHC8. Indeed, ABCA1 and DHHC8 immunoprecipitate together, indicating that these proteins are able to interact with each other (Figure 7C), fulfilling a feature of PAT/substrate specificity.
We show here that ABCA1 is robustly palmitoylated and that the palmitoylation of ABCA1 regulates its localization at the plasma membrane and contributes to its efflux function. The palmitoylation of ABCA1 is mediated in the most part by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux.
ABCA1 localization at the plasma membrane is considered essential for its ability to transport lipids across membranes. How ABCA1 is targeted to membranes was unclear. Palmitoylation regulates the localization of many proteins and is the only reversible lipid modification. As such, palmitoylation is dynamically regulated and plays an essential role in the nervous system, regulating aspects of neurotransmission, a process requiring exquisite regulation.25 We hypothesized that palmitoylation of ABCA1 was a contributor to its subcellular targeting, because regulation of lipid transport is vital for cellular processes. Indeed, ABCA1 is palmitoylated at 4 cysteines, and we found that palmitoylation of ABCA1 is essential for its proper localization at the plasma membrane.
Although mutation of each of the cysteines reduced palmitoylation moderately, the mutation of all 4 cysteines reduced most of the ABCA1 palmitoylation. However, the magnitude of reduction in lipid efflux remained the same regardless of the level of reduction in palmitoylation. One possibility for this is that the alterations of each of the cysteines to serines resulted in altered ABCA1 conformation, causing changes in efflux independent of palmitoylation. This possibility is unlikely, firstly, because treatment of ABCA1-expressing cells with 2BP, the nonfunctional analog of palmitate also resulted in decreased efflux ostensibly without altering ABCA1 conformation. Secondly, each of the mutants still retains ≈50% efflux activity, including the quadruple mutant, which would be expected to show very little efflux resulting from the most severe disruption of its 3D structure. However, each of the single mutations, as well as the quadruple mutation, resulted in a similar loss of ABCA1 localization at the plasma membrane. Thus, a similar deficit in efflux ability in each of the mutants would be expected, resulting from the absence of each mutant at the plasma membrane. Thus, palmitoylation at each of the 4 cysteines is essential for the proper localization of ABCA1 at the plasma membrane.
Regardless of almost absent ABCA1 at the plasma membrane, a reduction in lipid efflux of only ≈50% is observed. Thus, our study provides further evidence for the fact that in addition to the direct lipidation of ApoA-I by ABCA1 at the plasma membrane, ABCA1 also contributes to the lipidation of ApoA-I using intracellular lipid pools. Indeed, as in the study using PEST-domain deleted ABCA1, which showed that ABCA1 at the plasma membrane contributes to ≈50% lipid efflux,12 our study also finds that removing plasma membrane ABCA1 reduces lipid efflux by ≈50%.
In addition, this finding indicates that palmitoylation of ABCA1 is essential for its localization at the plasma membrane and not for its localization in intracellular sites. As controls for the efflux assay, we used a cell line harboring M1091T, a naturally occurring patient mutation that was previously characterized as severe loss-of-function mutation8 and showed >80% reduction in efflux, likely representing the absence of ABCA1 both at the plasma membrane and at intracellular sites of function.
ABCA1 is palmitoylated at 4 different cysteine residues. This in itself is not unusual because many proteins contain multiple sites for palmitoylation. The palmitoylation at different sites on a protein can either signify different functional outcomes depending on which cysteine is modified or the same functional role regardless of which cysteine is palmitoylated. Examples of each have been described. Regulator of G protein signaling (RGS)4, a member of a protein family that has GTPase activity protein activity is palmitoylated at 3 cysteine residues.28 The addition of palmitate to C2 and C12 is required for RGS4 localization to membrane raft domains. Palmitoylation of the C95 internal residue is required for RGS4 GTPase activity protein activity. Thus, RGS4 is a protein where palmitoylation at different residues modulates different functions. Palmitoylation of SNAP-25 (synaptosomal-associated protein of 25 kDa) occurs at 4 sites. Unlike RGS4, palmitoylation at all 4 cysteines shares the singular aim of increasing membrane association of SNAP-25.29 ABCA1 palmitoylation appears to follow the latter model where palmitoylation at all cysteines lead to ABCA1 localization at the plasma membrane.
Of the 23 mammalian DHHC domain–containing palmitoyl transferases catalyzing the addition of palmitate, DHHC8 increased ABCA1 palmitoylation most significantly. DHHC8 transcripts are expressed ubiquitously.26 Our finding that increasing DHHC8 levels increases ABCA1-mediated lipid efflux also suggests that raising ABCA1 palmitoylation through raising DHHC8 activity may be a viable therapeutic strategy for raising plasma HDL-C. Transgenic mice overexpressing DHHC8 would be valuable to confirm the role of this PAT in cholesterol metabolism. In addition, identification of the thioesterase that mediates the removal of palmitate from ABCA1 would aid in the development of therapeutics aimed at inhibiting removal of palmitate on ABCA1. Thus far, much study has focused on therapeutics that increase ABCA1 levels transcriptionally and have been fraught with difficulty because of untoward off target side effects. Thus, raising ABCA1 palmitoylation and therefore its function represents a potential new target for therapeutic development.
Together, our findings show that ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111 and that this palmitoylation is modulated by the palmitoyl transferase DHHC8. We also find that palmitoylation of ABCA1 is essential for its proper plasma membrane localization and efflux function. How ABCA1 transport is regulated was previously unknown. We have shown here that palmitoylation is essential to this process.
Sources of Funding
This work was supported by the Canadian Institutes for Health Research (MRH), a Grant-in-Aid from the Heart and Stroke Foundation of BC and Yukon (MRH), the Heart and Stroke Foundation of BC and Yukon (RRS), the Michael Smith Foundation for Health Research (RRS) and a Pacific Century Graduate Scholarship and University Graduate Fellowship (MHK). MRH holds a University Killam Professorship and a Canada Research Chair in Human Genetics.
Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, Loubser O, Ouelette BF, Fichter K, Ashbourne-Excoffon KJ, Sensen CW, Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S, Kastelein JJ, Genest J Jr, Hayden MR. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Gen. 1999; 22: 336–345.
Bodzioch M, Orsó E, Klucken J, Langmann T, Böttcher A, Diederich W, Drobnik W, Barlage S, Büchler C, Porsch-Özcürümez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, Schmitz G. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier Disease. Nat Genet. 1999; 22: 347–351.
Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M, Blanchette-Mackie J, Santamarina-Fojo S, Brewer HB Jr. Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem. 2001; 276: 27584–27590.
Chisholm JW, Burleson ER, Shelness GS, Parks JS. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL. J Lipid Res. 2002; 43: 36–44.
Chen W, Sun Y, Welch C, Gorelik A, Leventhal AR, Tabas I, Tall AR. Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes. J Biol Chem. 2001; 276: 43564–43569.
Singaraja RR, Visscher H, James ER, Chroni A, Coutinho JM, Brunham LR, Kang MH, Zannis VI, Chimini G, Hayden MR. Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro. Circ Res. 2006; 99: 389–397.
Vedhachalam C, Ghering AB, Davidson WS, Lund-Katz S, Rothblat GH, Phillips MC. ABCA1-induced cell surface binding sites for ApoA-I. Arterioscler Thromb Vasc Biol. 2007; 1603–9.
Witting SR, Maiorano JN, Davidson WS. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1. J Biol Chem. 2003; 278: 40121–40127.
Neufeld EB, Stonik JA, Demosky SJ Jr, Knapper CL, Combs CA, Cooney A, Comly M, Dwyer N, Blanchette-Mackie J, Remaley AT, Santamarina-Fojo S, Brewer HB Jr. The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease. J Biol Chem. 2004; 279: 15571–15578.
Chen W, Wang N, Tall AR. A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes. J Biol Chem. 2005; 280: 29277–29281.
Fitzgerald ML, Mendez AJ, Moore KJ, Andersson LP, Panjeton HA, Freeman MW. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein’s first hydrophilic domain to the exoplasmic space. J Biol Chem. 2001; 276: 15137–45.
Wellington CL, Singaraja R, Ellerby L, Savill J, Roy S, Leavitt B, Cattaneo E, Hackam A, Sharp A, Thornberry N, Nicholson DW, Bredesen DE, Hayden MR. Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J Biol Chem. 2000; 275: 19831–19838.
Wellington CL, Walker EK, Suarez A, Kwok A, Bissada N, Singaraja R, Yang Y-Z, Zhang LH, James E, Wilson JE, Francone O, McManus BM and Hayden MR. ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation. Lab Invest. 2002; 82: 273–283.
Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M, Mullard A, Haigh B, Gauthier-Campbell C, Gutekunst CA, Hayden MR, El-Husseini A. Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins. Neuron. 2004; 16: 44: 901–902.
Kaufman JF, Krangel MS, Strominger JL. Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds. J Biol Chem. 1984; 259: 7230–7238.
Tu Y, Popov S, Slaughter C, Ross EM. Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10. J Biol Chem. 1999; 274: 38260–38267.