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Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Centre, University of Amsterdam, The Netherlands
1Correspondence: Lab Genetic Metabolic Diseases, Rm. F0-226, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: c.vanroermund{at}amc.uva.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.—van Roermund, C. W. T., Visser, W. F., IJlst, L., van Cruchten, A., Boek, M., Kulik, W., Waterham, H. R., Wanders, R. J. A. The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl-CoA esters.
Key Words: β-oxidation • ATP-binding-cassette transporter • FA transport • carrier
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The importance of peroxisomes in fatty acid β-oxidation is emphasized by the existence of a number of different genetic diseases in which peroxisomal fatty acid β-oxidation is impaired (2)
. One of these peroxisomal disorders is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene (3)
. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family, is located in the peroxisomal membrane (4
5
6
7
8)
, and is a half transporter, rather than a full transporter like cystic fibrosis transmembrane conductance regulator. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), which is due to the impaired peroxisomal β-oxidation of these fatty acids. Although this suggests that ALDP functions in VLCFA import, no conclusive experimental evidence is available that support this notion (8)
.
To study the transport of fatty acids across the peroxisomal membrane and their metabolism, we chose Saccharomyces cerevisiae as a model system (9)
. An important advantage of such studies is that, at least in yeast, peroxisomes are the only organelles in which β-oxidation of fatty acids takes place in contrast to the situation in mammalian cells. In the latter case, peroxisomes as well as mitochondria participate in fatty acid oxidation (10)
. Earlier studies (11
12
13)
performed in the yeast S. cerevisiae have shown that in vivo peroxisomes are closed structures, which implies the existence of peroxisomal transport systems for metabolites including fatty acids. Candidate transporter proteins involved in peroxisomal fatty acid transport are Pxa1p and Pxa2p, which are the two yeast orthologs of the four mammalian ABC half transporters, named ALDP (ABCD1) (3)
, ALDR (ABCD2) (14)
, PMP70 (ABCD3) (15)
, and PMP69 (ABCD4) (16)
encoded by the ABCD1, ABCD2, ABCD3, and ABCD4 genes, respectively (17)
.
Previously, two independent pathways for fatty acid transport across the peroxisomal membrane have been identified in S. cerevisiae. The first pathway preferentially accepts long-chain fatty acids (LCFAs) and is dependent on the peroxisomal ABC transporters Pxa1p and Pxa2p, which have been claimed to act as acyl-CoA ester transporters (18)
. The other appears more specific for medium-chain fatty acids (MCFAs) and is dependent on the intraperoxisomal acyl-CoA synthetase Faa2p (see Fig. 1
).
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Far less is known about the function of mammalian peroxisomal ABC half transporters. For example, it remains to be established whether they function as homo- or heterodimers and whether they transport acyl-CoA esters across the peroxisomal membrane and, if so, which types of acyl-CoA species (11
, 19)
. To address these issues, we studied the properties of both the yeast and human peroxisomal ABC transporters. To this end, we measured the β-oxidation of different fatty acids in intact yeast cells expressing the different transporters. Furthermore, we determined the acyl-CoA profiles in these cells, using a novel tandem-mass spectrometry (MS/MS) based assay. Using this latter method, we found evidence for a role of the peroxisomal ABC transporters Pxa1p and Pxa2p in the peroxisomal import of acyl-CoA esters.
Interestingly, the mutant phenotype of the pxa1/pxa2 mutant, i.e., impaired growth in oleate containing medium and deficient oxidation of oleic acid, could be rescued, at least partially, by the expression of the human ABCD1 cDNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, leu2, trp1, ura3-251, prb1-1122, pep4-3, and gal2). The β-oxidation mutants used in this were as follows: pox1 or fox1 (deletion of acyl-CoA oxidase), fox2 (deletion of multifunctional protein), pot1 or fox3 (deletion of 3-ketoacyl-CoA thiolase), pxa1 and pxa2 (deletion of the peroxisomal ABC transporters 1 and 2), and faa2 (deletion of acyl-CoA synthetase). The mutants were constructed from BJ1991 as described previously (19)
. Double and triple mutants as used in this study were pxa1/pxa2, faa2/pxa1, faa2/pxa2, pxa1/fox2, pxa2/fox2, faa2/fox2, and faa2/pxa2/fox2 deletion strains. Yeast transformants containing the expression plasmids pALDP (human ABCD1), pPxa1p (yeast PXA1), and pPxa2p (yeast PXA2) were selected and grown in minimal medium containing 6.7 g/L yeast nitrogen base without amino acids (YNB-WO), supplemented with 3 g/L glucose and amino acids (20 mg/L) if required. For the induction of peroxisome proliferation, cells were shifted to YPO medium containing 5 g/L potassium phosphate buffer (pH 6.0), 3 g/L yeast extract, 5 g/L peptone supplemented with 1.2 g/L oleate, and 2 g/L Tween-80. Before being shifted to these media, the cells were grown in minimal 3 g/L glucose medium for at least 24 h.
Construction of ABCD1-expression plasmids
We used the human ABCD1 cDNA as a template to polymerase chain reaction (PCR) amplify two DNA fragments containing a 5'-part and a 3' part of ABCD1. The four primers used to amplify the 5' and 3' ABCD1 fragments (883 and 1389 bp, respectively) were 1ALDfHindIII (forward: 5'part ABCD1) 5'-TTTAAGCTTATGCCAGTTTTGTCTAGACCTAGACCATGGAGAGGTAACACGCTGAAGCGCACG-3' and (reverse: 5'part ABCD1) 5'-CTCCGAATTCGCCACCACACGCGAGTGCATGTAGC-3'. The second primer set was 860ALDf-EcoRI (forward: 3'part ABCD1): TGGCGAATTCGGAGGAGATCGCCTTCTATGG-3' and 5'-TTTTGTCGACTCAGGTGGAGGCACCCTG-3' (reverse: 3'part ABCD1). The 1ALDfHindIII primer was designed in such a way that the first 11 codons were optimized for the preferred codon usage of S. cerevisiae. The full-length ABCD1 open reading frame (ORF) was constructed by the EcoRI digestion of both amplicons followed by ligation of the EcoRI site of the 5' and 3' parts together. Next, ABCD1 was subcloned into the expression vectors that contained the oleate-inducible catalase promoter (Pca31) (20)
. All PCR fragments were sequenced to ensure that the sequences contained no PCR-introduced errors.
Acyl-CoA measurements
Samples of 20 mg of freeze-dried oleate-grown yeast cells were transferred to 1.5 ml Eppendorf vials. Exact sample weights were determined using a microbalance. Twenty microliters of 100 µM heptadecanoyl-CoA (C17-CoA) in 70% acetonitrile was added as internal standard, followed by 1.5 ml Doles reagent, i.e., heptane:isopropanol:H2SO4 (0.5 M),1:1:0.1 (v/v/v). The suspension was sonicated twice for 20 s at 2.5 W with a probe-tip vibra-cell sonicater (Sonics & Materials, Danburry, CT, USA), while cooling in ice water. Successively, 0.7 ml ammonium acetate (40 mM, pH=6.0), 100 µl BSA (1 mg/ml), and 1 ml heptane were added. After 3 min of mixing, samples were centrifuged at 1600 g (5 min, 4°C); 1.5 ml of the lower layer was collected, transferred to a glass tube, and evaporated to dryness (N2, 40°C). The final residue was reconstituted in 100 µl acetonitrile/water, at 7:3 (v/v). For the determination of the recovery of the internal standards, samples of four yeast cultures were prepared with varying amounts of internal standard (C17-CoA) using the same extraction procedure as described above. The recovery of the internal standards was considered to be a measure for the recovery of the endogenous compounds. The variation within one yeast culture was determined by analyzing the acyl-CoAs levels in four different cell cultures.
The acyl-CoAs were quantified by HPLC-MS/MS using electrospray ionization. The HPLC system consisted of a Surveyor MS-pump with degasser and a Surveyor autosampler with column oven (Thermo Finnigan Corporation, San Jose, CA, USA). Ten microliters of the extract was injected for analysis onto a YMC-Pack Pro C4 column (2.1x100 mm, YMC Europe, Dinslaken, Germany). Samples were eluted with a flow rate of 250 µl/min using a linear gradient of solution A, 50 mM ammonium acetate (pH=7.0); solution B, 100% acetonitrile; and solution C, 20 mM ammonium bicarbonate. The chromatographic gradient was programmed as follows: 0–5 min, 100% A to 30% B + 70% A; 5–5.1 min, 30% B + 70% C; 5.1–10.1 min, 70% B + 30% C; 10.1–15 min, isocratic 70% B + 30% C; and 15–15.1 min, 100% A; equilibration time with solvent A of 5 min. Column temperature was kept at 40°C.
MS/MS analyses were performed on a TSQ Quantum AM (Thermo Finnigan) operated in the negative ion mode. The source-induced dissociation (SID) was set at 20 V, spray voltage was 2500 V, and capillary temperature was 300°C. Argon was used as collision gas at a pressure of 1.5 mTorr, collision energy was 30 eV for the optimized transitions in the multiple reaction monitoring mode: m/z 508.5 
m/z 79.0 for C17-CoA (internal standard), m/z 514.5 m/z 79.0 for C18:1-CoA, and m/z 513.5 m/z 79.0 for C18:2-CoA.
Subcellular fractionation and Nycodenz gradients
Subcellular fractionation was performed as described by Van der Leij et al. (21)
. Organellar pellets were layered on top of a 15–35% (w/v) Nycodenz gradient (12 ml), with a cushion of 1.0 ml of 50% (w/v) Nycodenz solution. All Nycodenz solutions contained 5 mM morpholinoethanesulfonic acid (MES; pH 6.0), 1 mM EDTA, 1 mM KCl, and 8.5% (w/v) sucrose. The sealed tubes were centrifuged for 2.5 h in a vertical rotor (MSE 8x35) at 19,000 rpm at 4°C. After centrifugation, gradients were aliquoted in 12 fractions, which were assayed for activity of various marker enzymes as described below. In addition, 150 µl aliquots were taken from the individual fractions derived each from Nycodenz gradient, to which 150 µl of Laemmli sample buffer was added followed by analysis by SDS-PAGE.
Western blotting
Yeast extracts were subjected to SDS-PAGE on 10% acrylamide gel. ALDP was visualized using a monoclonal antibody (1:1500) following the manufacturer’s instructions (Euromedex, Souffelweyersheim, France).
Enzyme assays
β-Oxidation assays in intact cells were performed as described previously by van Roermund et al. (20)
, with some modifications. Cells were grown overnight in media containing oleate to induce fatty acid β-oxidation. The β-oxidation capacity was measured in 50 mM MES (pH=6.0) supplemented with 10 µM of [1-14C]-fatty acids. Subsequently, [14C]CO2 was trapped in 2 M NaOH and used to quantify the rate of fatty acid oxidation. Results are presented in precent relative to the rate of oxidation of wild-type cells grown on oleate. Rates of octanoic (C8:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), oleic (C18:1), linoleic (C18:2), behenic (C22:0), and tetracosahexaenoic acid (C24:6) were 7.84 ± 0.9, 7.24 ± 0.8, 1.5 ± 0.2, 1.06 ± 0.1, 2.6 ± 0.5, 2.73 ± 0.51, 0.15 ± 0.008, and 0.31 ± 0.06 nmol/min/OD, respectively.
The activity of the peroxisomal marker 3-hydroxyacyl-CoA dehydrogenase (3HAD) was measured on a Cobas-Fara centrifugal analyzer (Roche Diagnostics, Basel, Switzerland) by monitoring the acetoacetyl-CoA-dependent rate of NADH consumption at 340 nm (22)
. Fumarase activity was measured on a Cobas-Fara centrifugal analyzer monitoring the APADH production at 365 nm (23)
. The reaction was started with 10 mM fumarate in an incubation mixture of 100 mM Tris (pH 9.0), 1 g/L Triton X-100, 4 U/ml malate dehydrogenase, and 1 mM APAD for 5 min at 37°C. Protein concentrations were determined by the bicinchoninic acid method described by Smith et al. (24)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To study which of these two routes participates in the oxidation of other fatty acids, we performed whole-cell β-oxidation activity measurements in wild-type, pxa1, pxa2, and pxa1/pxa2 cells, using [1-14C]-labeled fatty acids of different chain length (Table 1
). The results show that the oxidation of MCFAs like octanoic (C8:0) and lauric (C12:0) acid was only slightly reduced in pxa1, pxa2, and pxa1/pxa2 cells. Oxidation of LCFAs like oleic (C18:1) and linoleic (C18:2) acid or VLCFAs like tetracosahexaenoic (C24:6) acid, however, was 
50% in these cells. The finding that MCFA β-oxidation was completely normal in pxa1, pxa2, and pxa1/pxa2 cells indicates that the actual β-oxidation machinery per se was not affected and that the peroxisomal induction was normal in these mutant cells. Taken together, the Pxa1p/Pxa2p heterodimer is required for the oxidation of a variety of different LCFAs and VLCFAs.
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Pxa1p and Pxa2p are involved in the transport of a broad spectrum of acyl-CoA esters across the peroxisomal membrane
To determine whether the Pxa1p/Pxa2p heterodimer is also capable of transporting MCFAs across the peroxisomal membrane, we repeated the β-oxidation activity measurements of Table 1
with myristic acid as a substrate in a mutant lacking the peroxisomal Faa2p, but containing a cytosolic version of Faa2p. This mutant was previously described by Hettema et al. (19)
and activates cytosolic MCFAs into their corresponding CoA esters. The results in Fig. 2
show that the deficient oxidation of myristic acid in faa2 cells was restored in faa2 cells in which the cytosolic version of Faa2p was introduced. Disruption of PXA1 or PXA2 in this faa2.pFaa2p(cytosol) mutant, however, led to a full block in myristic acid β-oxidation again, which confirms that not the substrate, i.e., myristate, but the product of the Faa2p reaction in the cytosol, i.e., myristoyl-CoA, is the substrate for the Pxa1p/Pxa2p heterodimer. In general, this implies that the route taken by a particular fatty acid is dependent on the subcellular site of activation to a CoA ester, i.e., cytosolic vs. intraperoxisomal in case of the experiment of Fig. 2
.
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To obtain additional evidence for the notion that the Pxa1p/Pxa2p heterodimer transports long-chain acyl-CoA esters across the peroxisomal membrane, we determined acyl-CoA profiles under in vivo conditions in different transformed yeast cells. To achieve this goal, we first developed a method for the quantification of acyl-CoA esters using HPLC-MS/MS (see Materials and Methods). This method was subsequently used to determine the oleoyl-CoA levels in wild-type and mutant cells. Figure 3A
shows that oleoyl-CoA levels were increased in the fox1 mutant in which the gene coding for acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation machinery in S. cerevisiae, is disrupted. Similar results were found in the fox2 and fox3 mutants. Interestingly, oleoyl-CoA levels were also elevated in the pxa1, pxa2, and pxa1/pxa2 disruption mutants, despite the fact that the β-oxidation of oleoyl-CoA was normal when measured in lysates of pxa1, pxa2, and pxa1/pxa2 cells (19)
. The most logical explanation for these data is that oleoyl-CoA accumulates in the cytosolic compartment due to a block in the uptake into peroxisomes. This will prevent the oleoyl-CoA from becoming a substrate for the intraperoxisomal β-oxidation machinery. These results are fully in line with a role of Pxa1p/Pxa2p in the transport of acyl-CoA esters. As a consequence, one would expect lower ratio of the C18:2CoA/C18:1CoA in the pxa1, pxa2, and pxa1/pxa2 cells (Fig. 3B
).
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As a positive control, we included the fox2 mutant in which the C18:2-CoA, which is the ,β- unsaturated species of oleoyl-CoA as formed by Fox1p, but cannot be degraded. In this mutant, C18:2-CoA levels as well as the C18:2-CoA/C18:1-CoA ratios (Fig. 3C
) were indeed higher when compared with wild-type cells.
Because C18:2-CoA can only be formed from oleoyl-CoA in the peroxisomal matrix via Fox1p, measurement of C18:2-CoA in addition to C18:1-CoA gives information on whether oleoyl-CoA is able to enter the peroxisomal matrix or not. Indeed, if Pxa1p/Pxa2p would be involved in the transport of oleoyl-CoA across the peroxisomal membrane, one would expect the C18:2-CoA/C18:1-CoA ratios to be lower in the pxa1/fox2 and pxa2/fox2 double mutants, as well as in the pxa2/faa2/fox2 triple mutant, in comparison with the fox2 single mutant, which is exactly what we found experimentally (Fig. 3C
).
Restoration of LCFA β-oxidation in pxa1/pxa2 cells by human ALDP
To be able to study the properties of human ALDP in comparison with Pxa1p and/or Pxa2p, we expressed human ALDP from an oleate inducible promoter in S. cerevisiae. Immunoblot analysis of ALDP using the cells transformed with human ABCD1 confirmed proper expression (Fig. 4
). Fractionation of a homogenate prepared from ALDP-expressing wild-type cells grown on oleate showed that ALDP was present in the crude organellar pellet (Fig. 4A
). Subsequent fractionation of the organellar pellet by equilibrium density gradient centrifugation showed that ALDP cofractionated with the peroxisomal matrix marker 3HAD but not with the mitochondrial marker (Fig. 4B
), indicating that the human ALDP expressed in S. cerevisiae is localized correctly in peroxisomes.
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Interestingly pxa1/pxa2 cells transformed with human ALDP showed partial restoration of growth on oleate (Fig. 5A
) and oxidation of oleate (Fig. 5B
) when compared with nontransformed pxa1/pxa2 cells. As a positive control, we also transformed PXA1 and/or PXA2 to the pxa1/pxa2 double mutant. The β-oxidation was only restored if both PXA1 and PXA2 were coexpressed (Fig. 6
). Surprisingly, no restoration of growth on oleate (Fig. 5A
) or oleate β-oxidation activity (Fig. 5B
) was observed when ABCD1 was coexpressed together with the yeast homologue PXA1, which could be due to the formation of an inactive dimer of these two half transporters under these conditions.
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ALDP can function as a homodimer and accepts a range of acyl-CoA esters
To study whether the mammalian peroxisomal ABC half transporter ALDP is involved in the transport of acyl-CoA esters like the yeast orthologs Pxa1p and Pxa2p, we used the same experimental setup as outlined above. Figure 3B
shows that introduction of human ABCD1 into pxa1/pxa2 cells had a marked effect on the C18:2-CoA/C18:1-CoA ratio, which indicates that the ALDP homodimer is also able to mediate the uptake of acyl-CoAs across the peroxisomal membrane.
To study the substrate specificity of ALDP in the experimental system described above, we performed whole-cell fatty acid β-oxidation activity measurements with a number of different substrates, including octanoic (C8:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), oleic (C18:1), linoleic (C18:2), behenic (C22:0), and tetracosahexaenoic acid (C24:6) in pxa1/pxa2 cells, transformed with human pALDP or not. Figure 7
shows that the rates of C18:1, C16:0, C22:0, and C24:6 β-oxidation were higher in pxa1/pxa2 cells transformed with ABCD1 when compared with the mutant transformed with an empty vector.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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indicated the existence of two routes of fatty acid uptake into peroxisomes, one taken by MCFAs and the other by LCFAs. The first pathway involves the transport of free fatty acids across the peroxisomal membrane followed by activation of the corresponding CoA esters by the intraperoxisomal enzyme Faa2p and subsequent β-oxidation, whereas in the other pathway fatty acids undergo extraperoxisomal activation followed by import of the acyl-CoA esters via Pxa1p/Pxa2p followed by β-oxidation. The ultimate proof for the postulate that the Pxa1p/Pxa2p heterodimer is able to transport acyl-CoA esters has to come from in vitro transport assays, preferably using purified proteins isolated from peroxisomal membranes or after expression in a suitable expression system, for instance as His-tagged protein. Unfortunately, this strategy has proven unsuccessful so far, both in our own hands and those of others (25)
. In this study, we have used a different strategy to shed light on the nature of the substrates used by the yeast Pxa1p/Pxa2p heterodimer, which is based on the analysis of acyl-CoA esters in both wild-type and mutant cells.
The first issue addressed in this study was whether oleoyl-CoA accumulates in pxa1, pxa2, and pxa1/pxa2 mutants. Resolution of this issue is of utmost importance, since so far all the conclusions drawn by Hettema et al. (19)
were only extrapolated from the finding that overall oleate β-oxidation was deficient in these different mutants. The results of Fig. 3A
show that oleoyl-CoA accumulates in the same way as in the mutants in which the different β-oxidation enzymes per se were disrupted. Since the activity of the β-oxidation enzymes, including Fox1p, Fox2p, and Fox3p, is normal in pxa1, pxa2, and pxa1/pxa2 mutants, as demonstrated also by the fact that oleate β-oxidation is normal in lysates of these mutants, these results strongly suggest that the oleoyl-CoA, which accumulates in the latter mutants, is physically separated from the β-oxidation enzymes and therefore probably present in the cytosol. Our finding that the levels of the β-oxidation intermediates, notably ,β-unsaturated oleoyl-CoA (i.e., C18:2-CoA), were actually lower in the pxa1/pxa2/fox2 mutant as compared with the levels in the fox2 mutant provides strong support for this conclusion. Taken together, in the absence of any direct evidence in which the transport of acyl-CoA esters by Pxa1p/Pxa2p is actually shown directly, our data strongly indicate that the Pxa1/Pxa2p heterodimer is indeed transporting acyl-CoA esters.
The results described in this study also show that the function of Pxa1p/Pxa2p can be taken over by human ALDP, at least in part, as concluded from the fact that growth on oleate is partially restored on expression of ABCD1 in pxa1/pxa2 cells. Furthermore, the acyl-CoA ester profile was partially corrected on expression of ABCD1 in pxa1/pxa2 cells.
In X-linked adrenoleukodystrophy, in which ABCD1 is mutated, there is accumulation of C24:0 and especially C26:0, whereas the levels of other fatty acids, including C18:1, are normal. Unfortunately, the oxidation rate of C26:0 is extremely low in S. cerevisiae, whether human ABCD1 is present or not. To be able to study the function of ALDP in the transport of these CoA esters, using S. cerevisiae as model system, we will need to study which enzyme and/or transport step limits C26:0 β-oxidation in S. cerevisiae. One reason may be that the actual β-oxidation machinery per se is not active with C26:0-CoA or that the capacity of S. cerevisiae to activate these VLCFAs is limiting. These problems can possibly be overcome by expression of the human peroxisomal β-oxidation enzymes involved in C26:0 β-oxidation. The activation of C26:0 may also be limiting for C26:0 β-oxidation in S. cerevisiae. In this respect, recent work by Jia et al. (26)
has to be mentioned, as it has clearly shown that FAT4p is the predominant enzyme catalyzing the activation of VLCFAs at least when β-oxidation is concerned. In summary, we conclude that the human peroxisomal ABC transporter ALDP functions as a homodimer and accepts a range of acyl-CoA esters.
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ACKNOWLEDGMENTS |
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Received for publication April 30, 2008. Accepted for publication July 24, 2008.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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