Biochemical and Biophysical Research Communications
Characterization of human ATP-binding cassette protein subfamily D reconstituted into proteoliposomes
Introduction
ATP-binding cassette (ABC) proteins are a large family of membrane-bound proteins with highly conserved structure. In humans, there are 48 members, classified into seven subfamilies, A to G, based on structural organization and amino acid homology [1,2]. These proteins catalyze the ATP-dependent transmembrane transport of a wide variety of substrates, and defects in their function are involved in various inherited metabolic diseases.
To date, four ABC proteins have been included in subfamily D (ABCD) proteins in mammals [3]. They are half-sized ABC proteins and exist mainly as a homodimer [4]. ABCD1‒3 are located on peroxisomal membranes. ABCD1 and 2 play an important role in the transport into peroxisomes of very long chain fatty acids (VLCFAs)-CoA with slightly different substrate specificity [5,6]. ABCD3 has been reported to be involved in the transport of branched chain fatty acid-CoA and di- and tri-hydroxycholestanoyl-CoA, which are intermediates of bile acid synthesis [7]. Dysfunction of ABCD1 and ABCD3 causes the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) and severe liver disease, respectively. VLCFA and the intermediates of bile acid accumulate in the tissues of these patients [8,9].
On the other hand, ABCD4 is located on lysosomal membranes [10]. ABCD4 barely interacts with the peroxisomal biogenesis factor Pex19p, because of its lack of NH2-terminal hydrophobic motif. As a result, the transmembrane domain (TMD) 1 of ABCD4 is recognized by certain signal recognition particles and integrated into the ER membrane [11]. ABCD4 dimer then makes a complex with lysosomal membrane protein LMBD1, an adopter protein, and targets to lysosome [12]. ABCD4 is thought to be involved in the transport of vitamin B12 from lysosomes to the cytosol. ABCD4 dysfunction results in a failure of lysosomal vitamin B12 release, suggesting that ABCD4 functions as a transporter of vitamin B12 from inside to outside lysosomes [10]. Interestingly, the transport direction of ABCD4 is opposite to that of ABCD1‒3 although they all share the same protein topology. Yet, the precise transport mechanism of these ABC transporters has yet to be adequately characterized.
In addition, a homolog of human ABCD1 in plants, COMATOSE (CTS), was recently found to have acyl-CoA thioesterase activity and VLCFA-CoA is suggested to be hydrolyzed prior to transport based on the finding that mutants with defect the activity had impaired fatty acid β-oxidation in plant cells [13]. On the other hand, it has been demonstrated that the β-oxidation of VLCFA-CoA in isolated peroxisomes from human fibroblasts is not stimulated by CoA, suggesting that VLCFA-CoA is transported directly [14]. Therefore, it is still controversial whether ABCD proteins translocate acyl-CoAs or free fatty acids, and precise characterization of the transport mechanism is required.
It was difficult to obtain enough amount of ABCD1‒4 for analyzing their functions even when these proteins were overexpressed in mammalian cells. Therefore, we expressed ABCD1‒4 in methylotrophic yeast Pichia pastoris with a strong methanol-inducible alcohol oxidase gene, AOX1, promoter. We established a procedure for purifying ABCD1‒4 and reconstituting the detergent-solubilized proteins into liposomes, then analyzed the ATPase and acyl-CoA thioesterase activities. ABCD1‒4 showed stable ATPase activity that was inhibited by AlF3. Equal levels of acyl-CoA thioesterase activity were detected in ABCD1‒4, suggesting that this proteoliposome technique is useful for characterizing how ABCD1‒4 transport the substrate into the peroxisomes or outside of lysosomes, as well as the role played by acyl-CoA thioesterase activity.
Section snippets
Yeast strains and media
P. pastoris SMD1168 was used as a host strain to express human ABCD proteins. The strains were grown in YPD (1% yeast extract, 2% peptone and 2% glucose), BM (0.5% yeast extract and 1% methanol) or SD (0.67% yeast nitrogen base without amino acids and 2% glucose) medium.
Construction of the human ABCD expression plasmids
The oligonucleotide primers used for PCR reactions are listed in supplementary Table S1. The plasmid for expressing human ABCD1 was constructed as follows. The human ABCD1 gene was amplified with primer sets Fw-ABCD1/Rv-ABCD1
Expression and purification of human ABCD1
The purification and reconstitution of hABCD1 into liposomes is an important step toward a detailed functional understanding of the transporter. A sufficient amount of purified protein with retained function is required for the analysis. We previously expressed hABCD1 and hABCD2 in Sf21 cells, but were unable to obtain sufficient amounts of biologically active proteins [17]. We thus expressed hABCD1 as a fusion protein with a histidine-tag (His-hABCD1) under the control of the strong,
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26460063), JSPS Core-to-Core Program, B. Asia-Africa Science Platforms, and the Sasakawa Scientific Research Grant from The Japan Science Society (27-427). We would like to thank Golden (www.goldenenglishediting.com) and Pacific Edit (http://www.pacificedit.com/) for editing and proofreading this manuscript.
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These authors contributed equally to this work.