Peroxisomal ATP-binding cassette transporters form mainly tetramers

Flore Geillon,Catherine Gondcaille,Quentin Raas,Alexandre M.M Dias,Delphine Pecqueur,Caroline Truntzer,Géraldine Lucchi,Patrick Ducoroy,Pierre Falson,Stéphane Savary,Doriane Trompier

doi:10.1074/jbc.m116.772806

Abstract

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

Highlights

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities
ABCD1- and ABCD2-particle floatation data were analyzed by considering the apparent size of the complexes based on the position of the chimeric ABCD2-ABCD2-EGFP protein marker and by considering the shift in peak positions obtained depending on the detergents
We demonstrate the presence of tetrameric assemblies of the peroxisomal ABCD1 and ABCD2 transporters in rodents, the scientific community has previously reported a dimeric status for peroxisomal ABCD proteins in yeast [37, 38], rodents, or humans (8, 19 –23)

Summary

Introduction

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. ABCD2, the closest homolog, shares the same substrate preference with ABCD1 for saturated fatty acids (FA) and monounsaturated FA (MUFA) but has a distinct substrate preference for shorter VLCFA and polyunsaturated FA (PUFA) [5, 8, 9]. ABCD3 has the broadest substrate specificity because it is involved in the transport of saturated FA, unsaturated FA, branched-chain FA, dicarboxylic FA, and C27 bile acid intermediates [10, 11]

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