J Hum Genet (2005) 50:99–105 DOI 10.1007/s10038-004-0226-4 ORIGINAL ARTICLE Carla P. Guimara˜es Æ Clara Sa´-Miranda Jorge E. Azevedo Probing substrate-induced conformational alterations in adrenoleukodystrophy protein by proteolysis Received: 22 November 2004 / Accepted: 14 December 2004 / Published online: 29 January 2005 Ó The Japan Society of Human Genetics and Springer-Verlag 2005 Abstract The adrenoleukodystrophy protein (ALDP) is a mum incidence of 1:21,000 in the male population half-ABC (ATP-binding cassette) transporter localized in (Bezman et al. 2001). It is a progressive neurodegener- the peroxisomal membrane. Dysfunction of this protein is ative disease affecting mainly the nervous system white the cause of the human genetic disorder X-linked adre- matter and the adrenal cortex. The clinical presentation noleukodystrophy (X-ALD), which is characterized by is extremely variable and, frequently, different pheno- accumulation of saturated, very-long-chain fatty acids types occur in the same family. Common to all the (VLCFAs). This observation suggests that ALDP is in- X-ALD phenotypes, however, is the accumulation of volved in the metabolism of these compounds. Whether saturated, unbranched, very-long-chain fatty acids ALDP transports VLCFAs or their derivatives across the (VLCFAs) in plasma and tissues of the patients (Moser peroxisomal membrane or some cofactors essential for the et al. 2001). efficient peroxisomal b-oxidation of these fatty acids is The gene responsible for X-ALD (ABCD1 gene) was still unknown. In this work, we used a protease-based identified in 1993 (Mosser et al. 1993). It encodes a half- approach to search for substrate-induced conformational ATP-binding cassette (ABC) transporter, the so-called alterations on ALDP. Our results suggest that ALDP is ALDP (or ABCD1). Half-ABC transporters are directly involved in the transport of long- and very-long- homodimeric or heterodimeric protein assemblies that chain acyl-CoAs across the peroxisomal membrane. couple ATP hydrolysis to the transport of substrates across biological membranes (Dean et al. 2001). Data Keywords ATP-binding cassette transporters Æ ABCD1 suggesting that ALDP from mouse liver is a homodi- transporter Æ Lipid transport Æ VLCFA metabolism Æ meric protein were recently described [see Discussion in X-linked adrenoleukodystrophy Æ ALDP function (Guimaraes et al. 2004)]. Several hypotheses have been forwarded to explain Abbreviations ABC: ATP-binding cassette Æ how dysfunction of ALDP leads to accumulation of ALDP: Adrenoleukodystrophy protein Æ VLCFA: VLCFAs in X-ALD [e.g., (McGuinness et al. 2003; Saturated very-long-chain fatty acid Æ X-ALD: X-linked Mosser et al. 1993)]. However, the possibility that adrenoleukodystrophy ALDP may play a direct role in the metabolism of these fatty acids is still most plausible [e.g., (Aubourg and Dubois-Dalcq 2000; Braiterman et al. 1998; Dubois- Dalcq et al. 1999; Kemp et al. 2001)]. The observations Introduction supporting this hypothesis are not limited to the fact that b-oxidation of VLCFAs is a peroxisomal event X-linked adrenoleukodystrophy (X-ALD) is the most (Lazarow and De Duve 1976) and that ALDP is a per- common inherited peroxisomal disorder with a mini- oxisomal membrane protein (Mosser et al. 1994). In- deed, a variety of genetic and biochemical studies on C. P. Guimara˜ es Æ C. Sa´-Miranda Æ J. E. Azevedo (&) ALDP-related peroxisomal ABC transporters from UNILIPE, Instituto de Biologia Molecular e Celular, other organisms have led to the same conclusion. For Rua do Campo Alegre, 823, 4150-180 Porto, Portugal example, disruption of Pxa1p and/or Pxa2p, the only E-mail: [email protected] Tel.: +351-2-26074900 two peroxisomal ABC transporters known in Saccha- Fax: +351-2-26099157 romyces cerevisiae, results in impaired growth of these C. P. Guimara˜ es Æ J. E. Azevedo mutants on long-chain fatty acids as a sole carbon Instituto de Cieˆ ncias Biome´dicas de Abel Salazar, source (Hettema et al. 1996; Shani and Valle 1996; Porto, Portugal Shani et al. 1995). Biochemical characterization of these 100 proteins demonstrated their involvement in the trans- frozen immediately in liquid nitrogen and stored at port of acyl-CoAs across the peroxisomal membrane À70°C. (Hettema et al. 1996; Verleur et al. 1997). More recently, a similar role was proposed for the Comatose/Ped3 gene product of Arabidopsis thaliana (Footitt et al. 2002; Protease treatment and extraction of membrane proteins Zolman et al. 2001). Obviously, all these observations do not prove that ALDP has a similar role in the metabo- An aliquot of 0.45 mg of placenta organelle protein was lism of VLCFAs, but they do provide a ground of incubated at 26°C for 1 h with 9 lg Factor Xa in 0.9 ml testable hypotheses. of buffer containing 0.22 M sucrose, 10 mM Tris-HCl, If it is assumed that ALDP is directly involved in the pH 8.0, 1 mM CaCl2, and 10 mM MgCl2. Endogenous b-oxidation of VLCFAs, and if we consider that the proteases were inhibited with 0.1 mg/ml PMSF (added subcellular compartment where these fatty acids are from a 50 mg/ml stock solution in ethanol) and 1:200 (v/ activated to their CoA thioesters is presently unknown v) mammalian protease inhibitor cocktail for 5 min on [for a review see (Moser et al. 2001)], then there are at least ice. Extraction of membranes with alkaline or low and three different mechanisms leading to the same out- high ionic strength solutions were performed essentially come: ALDP may be directly involved in the peroxisomal as described (Fujiki et al. 1982; Gouveia et al. 2000). import of (1) free VLCFAs, (2) very-long-chain acyl- CoAs, or (3) a cofactor necessary for the b-oxidation of VLCFAs. In this work, we have addressed the first two In vitro synthesis of radiolabeled ALDP and cleavage possibilities. Our results suggest that ALDP is involved in with Factor Xa the translocation of long- and very-long-chain acyl-CoAs across the peroxisomal membrane. The cDNA encoding the human full-length ALDP transporter was amplified by RT-PCR using the F1 and F8 oligonucleotides exactly as described previously Materials and methods (Guimaraes et al. 2001) and cloned into the pGEM-T easy vector (Promega). Subsequently, the cDNA was Materials inserted into the EcoRI site of pGEM-4 (Promega). Clones with ALDP cDNA in the correct orientation Factor Xa protease was obtained from New England were selected for further in vitro transcription expri- Biolabs (#P8010, lot 55). Acetyl-CoA and dodecanoyl- ments with the Sp6 RNA polymerase. The plasmid was CoA were purchased from Roche and Fluka, respec- linearized with HindIII endonuclease and transcribed tively. Docosanoyl-CoA and tetracosanoyl-CoA were according to the manufacturer’s instructions (Roche). synthesized by American Radiolabeled Chemicals. All 35S-labeled ALDP was synthesized using the translation the other reagents were from Sigma. kit Reticulocyte Type II (Roche) in the presence of 35S-methionine (specific activity >1,000 Ci/mmol; ICN Biomedicals). Subcellular fractionation of placenta One microliter of reticulocyte lysates containing 35S-labeled ALDP was added to 60 ll of buffer con- A human placenta was obtained from the local mater- taining 0.25 M sucrose, 40 mM Tris-HCl, pH 8.0, 2 mM nity hospital. An informed written consent was granted. CaCl2, and 0.2 M NaCl. Then, Factor Xa was added and The tissue was chilled on ice immediately after birth and the reaction extended over 1 h 30 min at 26°C. transported to the laboratory. The following procedures were carried out at 4°C: The placenta was washed with SEI buffer (0.25 M sucrose; 1 mM EDTA-NaOH, Preparation of lipids pH 7.4; and 5 mM imidazole-HCl, pH 7.4) and the adherent membranes were cut off. A portion of Stock solutions of acyl-CoA esters were prepared at a final approximately 60 g was cut into small pieces and concentration of 0.3 mM in CHCl3:CH3OH (1:1). These extensively washed in SEI buffer. The tissue was were dried under a stream of nitrogen and resuspended homogenized using a polytron (model PT10/35, Ki- with a-cyclodextrin (10 mg/ml in 20 mM Tris-HCl, nematica, Switzerland) in four volumes of SEI buffer pH 8.0) in order to obtain 1 mM of final concentration supplemented with 0.1 mg/ml PMSF and a mammalian lipid. The suspensions were sonicated for 1 h at room protease inhibitor cocktail (Sigma) at a 1:450 (v/v) final temperature in an ultrasonic water bath (Sonorex super dilution. An organelle fraction was prepared by differ- RK255H, Bandelin). After checking the pH, single-use ential centrifugation. Briefly, the homogenate was aliquots were stored at À70°C. It should be noted centrifuged at 300· g for 10 min at 4°C in the SS-34 that all efforts to solubilize docosanoyl-CoA and rotor (Sorvall, model RC-5B PLUS) and afterward at tetracosanoyl-CoA in aqueous buffer (e.g., by increasing 3,200· g for 10 min. Finally, the organelle fraction was the ratio of a-cyclodextrin/lipid, increasing the solubili- obtained by centrifugation at 20,000· g for 20 min and zation temperature, extending the sonication time, or resuspended in SEI buffer. Aliquots (20–30 mg/ml) were lowering the lipid concentration to half) were ineffective. 101 Suspensions of these two lipids were stored in aliquots at a biological membrane. Although our knowledge on the À70°C. In the experiment presented in Fig. 2c, dodeca- mechanism by which this process occurs is still limited, noic and eicosanoic acids were added directly from a data indicating that these proteins oscillate between 6.5 mM stock solution in methanol.