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Peroxisomal ATP-Binding Cassette Transporters Form Mainly Tetramers

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Peroxisomal ATP-Binding Cassette Transporters Form Mainly Tetramers Peroxisomal ATP-binding cassette transporters form mainly tetramers Flore Geillon, Catherine Gondcaille, Quentin Raas, Alexandre Dias, Delphine Pecqueur, Caroline Truntzer, Géraldine Lucchi, Patrick Ducoroy, Pierre Falson, Stéphane Savary, et al. To cite this version: Flore Geillon, Catherine Gondcaille, Quentin Raas, Alexandre Dias, Delphine Pecqueur, et al.. Per- oxisomal ATP-binding cassette transporters form mainly tetramers. Journal of Biological Chem- istry, American Society for Biochemistry and Molecular Biology, 2017, 292 (17), pp.6965-6977. 10.1074/jbc.M116.772806. hal-02329531 HAL Id: hal-02329531 https://hal.archives-ouvertes.fr/hal-02329531 Submitted on 23 Oct 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. cros ARTICLE Peroxisomal ATP-binding cassette transporters form mainly tetramers Received for publication, December 16, 2016, and in revised form, March 3, 2017 Published, Papers in Press, March 3, 2017, DOI 10.1074/jbc.M116.772806 Flore Geillon‡, Catherine Gondcaille‡, Quentin Raas‡, Alexandre M. M. Dias‡, Delphine Pecqueur§, Caroline Truntzer§, Géraldine Lucchi§, Patrick Ducoroy§, Pierre Falson¶, Stéphane Savary‡, and Doriane Trompier‡1 From the ‡Laboratoire Bio-PeroxIL EA7270 and §CLIPP-ICMUB, Universite´Bourgogne-Franche-Comté, 6 Bd Gabriel, 21000 Dijon, France and the ¶Drug Resistance and Membrane Proteins Team, Molecular Microbiology and Structural Biochemistry Laboratory, Institut de Biologie et Chimie des Protéines (IBCP), UMR5086 CNRS/Université Lyon 1, 7 Passage du Vercors, 69367 Lyon, France Edited by George M. Carman ABCD1 and its homolog ABCD2 are peroxisomal ATP-bind- mediates the import of saturated and monounsaturated very ing cassette (ABC) half-transporters of fatty acyl-CoAs with long-chain fatty acids (VLCFA) as CoA esters into the peroxi- both distinct and overlapping substrate specificities. Although some for their further degradation by ␤-oxidation (3–5). A it is established that ABC half-transporters have at least to defect in ABCD1 results in increased levels of VLCFA in tissues dimerize to generate a functional unit, functional equivalents of and plasma (4). Downloaded from tetramers (i.e. dimers of full-length transporters) have also been Two homologs of ABCD1 that belong to the same D subfam- reported. However, oligomerization of peroxisomal ABCD ily are expressed in the peroxisome: ABCD2 (6) and ABCD3, transporters is incompletely understood but is of potential sig- also called PMP70 (7). ABCD2, the closest homolog, shares the nificance because more complex oligomerization might lead to same substrate preference with ABCD1 for saturated fatty acids differences in substrate specificity. In this work, we have char- (FA) and monounsaturated FA (MUFA) but has a distinct sub- http://www.jbc.org/ acterized the quaternary structure of the ABCD1 and ABCD2 strate preference for shorter VLCFA and polyunsaturated FA proteins in the peroxisomal membrane. Using various biochem- (PUFA) (5, 8, 9). ABCD3 has the broadest substrate specificity ical approaches, we clearly demonstrate that both transporters because it is involved in the transport of saturated FA, unsatu- exist as both homo- and heterotetramers, with a predominance rated FA, branched-chain FA, dicarboxylic FA, and C27 bile of homotetramers. In addition to tetramers, some larger molec- acid intermediates (10, 11). ular ABCD assemblies were also found but represented only a at CNRS on October 23, 2019 The partial overlap in substrate specificity between ABCD1 minor fraction. By using quantitative co-immunoprecipitation and its homologs explains their ability, upon overexpression, to assays coupled with tandem mass spectrometry, we identified compensate for ABCD1 defect (12–14). However, the basal potential binding partners of ABCD2 involved in polyunsatu- expression levels of ABCD2 and ABCD3 are not sufficient to rated fatty-acid metabolism. Interestingly, we identified cal- allow the compensation mechanisms to be efficient enough in cium ATPases as ABCD2-binding partners, suggesting a role of X-ALD patients. This may be explained by the fact that ABCD2 ABCD2 in calcium signaling. In conclusion, we have shown here expression mirrors ABCD1 distribution in many cell types in that ABCD1 and its homolog ABCD2 exist mainly as homote- tramers in the peroxisomal membrane. different tissues (15) and by the fact that ABCD3, although ubiquitously expressed (16, 17), has mainly a distinct substrate specificity (10, 11) and mediates the transport of ABCD1 sub- strates with less efficiency (3). The ABCD1 transporter belongs to the D subfamily of Peroxisomal ABCD transporters are half-transporters con- the ATP-binding cassette (ABC)2 transporter family and is taining one nucleotide-binding domain (NBD) and one trans- encoded by the ABCD1 gene (1). A large number of mutations membrane domain as opposed to ABC full-length transporters in ABCD1 are responsible for the most common peroxisomal that contain two copies of each domain. It is therefore com- disorder, which is called X-linked adrenoleukodystrophy (X-ALD) (2). Expressed at the peroxisomal membrane, ABCD1 monly accepted that half-transporters have to dimerize to gen- erate a minimal functional unit. The expression pattern of per- oxisomal ABC transporters allows, in principle, the formation This work was supported by grants from the regional council of Burgundy and of all combinations of heterodimers. Indeed, ABCD3 is ubiqui- from the University of Burgundy. The authors declare that they have no tously expressed, and although ABCD2 expression mirrors conflicts of interest with the contents of this article. 1 To whom correspondence should be addressed. Tel.: 33-380396202; Fax: ABCD1 distribution in many cell types in different tissues, 33-380396250; E-mail: [email protected]. ABCD2 expression is highly inducible (18). Thus, upon stimu- 2 The abbreviations used are: ABC, ATP-binding cassette; DHA, docosa- lation, both proteins can be expressed in the same cell. Actually, hexaenoic acid; EGFP, enhanced green fluorescent protein; IP, immuno- precipitation; MUFA, monounsaturated fatty acid(s); NBD, nucleotide- the presence of both peroxisomal ABCD heterodimers and binding domain; PUFA, polyunsaturated fatty acids; VLCFA, very homodimers has been evidenced mainly by FRET and co-im- long-chain fatty acid(s); X-ALD, X-linked adrenoleukodystrophy; AMP-PNP, munoprecipitation (co-IP) assays (8, 19–23). In addition, 5Ј-adenylyl-␤,␥-imidodiphosphate; FA, fatty acid(s); DDM, dodecyl malto- pyranoside; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)pro- because chimeric proteins mimicking homo- and heterodimers pane-1,3-diol; HB, homogenization buffer; ACN, acetonitrile. of ABCD1 and ABCD2 are functionally active (24), one can J. Biol. Chem. (2017) 292(17) 6965–6977 6965 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Molecular assembly of ABCD proteins Figure 1. Calibration of velocity sucrose gradients with soluble and membrane protein markers. A, 5–30% (w/v) sucrose gradient linearity was checked by monitoring of sucrose concentration using a refractometer. Fractions 1–22 correspond to fractions from the top to the bottom of the gradient. B, calibration curve was drawn from the flotation of native soluble protein markers (t, thyroglobulin (669 kDa); cat, catalase (232 kDa); b, ␤-amylase (200 kDa); c, conalbumin (75 kDa); o, ovalbumin (44 kDa)) in parallel and equivalent sucrose gradients. C, ABCD2-ABCD2-EGFP (194 kDa) solubilized with 1% SDS, used as a membrane protein size marker, floated in fractions 9 and 10 (indicated by a gray bar) as monitored by densitometry analysis of immunoblotting performed with an anti-GFP antibody for all collected fractions. speculate that alternative dimerization of peroxisomal ABCD tively low (31), but the ABCD1 protein expression level is transporters could lead to different substrate specificity. sufficient to allow VLCFA ␤-oxidation measurements (8). At Downloaded from It is noteworthy that the level of molecular complexity for floating equilibrium, 22 fractions were collected from a 5–30% peroxisomal ABCD half-transporters might be underesti- (w/v) sucrose gradient, checked for linearity (Fig. 1A), and cal- mated. Indeed, other ABC half-transporters, such as ABCG2, ibrated with native soluble markers (Fig. 1B) and with a mem- exhibit a tetrameric organization consisting in four core brane protein marker (denatured ABCD2-ABCD2-EGFP; Fig. domains (25). Moreover, dimers of full-length transporters 1C) performed to ensure a more accurate interpretation of the http://www.jbc.org/ (also yielding a four-core domain organization) were observed results. Indeed, it should be noted that the sedimentation prop- for ABCA1 (26), ABCA3 (27), ABCC1 (28), and ABCC7 (29). erties of membrane proteins depend on the amount of mem- Therefore, it is tempting to speculate that the interactions brane lipids and detergent bound (size of the micelle). Thus, described within the peroxisomal ABCD subfamily occur ABCD1- and ABCD2-particle
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