Sterol Transfer by ABCG5 and ABCG8

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for ␤-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2⌬ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2⌬ mutant, and restored ␤-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.Peroxisomes perform a range of different functions, including ␤-oxidation of fatty acids (FA) 2 and synthesis and degradation of bioactive, lipid-derived molecules. Import of substrates for peroxisomal metabolism is mediated by ATP binding cassette (ABC) transporters belonging to subclass D (1, 2). ABC transporters are composed of a minimum of four functional domains: two transmembrane domains, involved in substrate binding and translocation, and two nucleotide binding domains (NBDs) that bind and hydrolyze ATP, providing energy for transport (3, 4). The domains may be fused into a single polypeptide, but are frequently expressed as half-size transporters composed of a transmembrane domain fused to an NBD, which hetero-or homodimerize to form a functional transporter. Bakers' yeast (Saccharomyces cerevisiae) contains two ABCD genes that encode half-size ABC proteins: Pxa1p (peroxisomal ABC-transporter 1), and Pxa2p (5-7). The single pxa1⌬ and pxa2⌬ deletion mutants are unable to grow on oleate (C18:1) as the sole carbon source and exhibit reduced ␤-oxidation of this long-chain FA. It has been proposed that Pxa1p and Pxa2p operate as a heterodimer to form a functional transporter (6,8,9), which has been shown by indirect evidence to be required for the peroxisomal transport of the C18:1-CoA, a long-chain acyl-CoA ester, but not for import of C8:0-CoA (10). In contrast, medium-chain FAs enter yeast peroxisomes as free acids independently of Pxa1p/Pxa2p, and are activated by peroxisomal acyl-CoA synthetase, Faa2p, prior to ␤-oxidation (6).The human ABCD transporter subfamily comprises four half-size members: adrenoleukodystrophy protein (ALDP), ALD-related protein, the 70-kDa peroxisomal membrane protein (PMP70), and PMP70-related protein (PMP70R/PMP69) (1, 2). Although...

Section: Discussionsupporting

confidence: 77%

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

Nyathi

Lousa

Roermund

et al. 2010

“…We showed previously that mutations in the Walker A motif that are predicted to interfere with ATP hydrolysis abolish sterol transport when introduced into NBD2 but do not impair sterol transport in vivo and sterol transfer in vitro when introduced into NBD1 (3,16). A major finding of this study is that NBD1, although functionally degenerate, is not dispensable for sterol transport.…”

Section: Discussionsupporting

confidence: 49%

“…We previously arbitrarily referred to the active NBD of G5G8 as NBD1 and the inactive NBD as NBD2 (3,16,17). In this work, we have switched the numbering of the NBDs to be consistent with terminology used to describe asymmetric full ABC transporters where the inactive ATPase is N-terminal to the active ATPase and is therefore designated NBD1 (9).…”

mentioning

confidence: 99%

Sequences in the Nonconsensus Nucleotide-binding Domain of ABCG5/ABCG8 Required for Sterol Transport

Wang

Grishin

Kinch

et al. 2011

Self Cite

ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity. ATP-binding cassette (ABC)3 transporters hydrolyze ATP to transport a wide variety of substrates across membranes (1). The members of the family share a common architecture that comprises two nucleotide-binding domains (NBDs), which mediate ATP binding and hydrolysis, and two transmembrane domains (TMDs), each of which typically contains six transmembrane helixes that together form a conduit through which the substrate is translocated across the membrane (2). The sequences of the TMDs are widely divergent among family members. In contrast, the NBDs share several characteristic motifs and are much more strongly conserved.Canonical sequences found in the NBDs of ABC transporters include the Walker A motif (GXXGXGKST, where X is any amino acid), which contacts the ␣-and ␤-phosphates of ATP; the Walker B motif (DE, where is a hydrophobic amino acid), which positions a hydrolytic water molecule; the D-loop (SALD), which may contribute to the dimer interface; and a five-residue C-loop (alternatively called the signature sequence; LSGGQ) that packs against the ␥-phosphate and closes off the active site (2). The NBDs also share conserved functional residues, including an A-loop aromatic residue that stacks with the adenine base of the bound nucleotide, a switch motif histidine that contacts the ␥-phosphate, and a Q-loop glutamine that plays a role in mediating communication between the NBDs and TMDs (2).Structural studies have indicated that the NBDs form dimers arranged in a head-to-tail orientation such that the Walker A and Walker B motifs of one NBD pair with the C-loop of the second NBD (2-7). In several ABC transporters, the two NBDs differ in ATP catalytic activity. These transporters invariably ...

“…Recent progress has been made using purified G5G8 complexes and demonstrates that sterols are indeed substrates for the G5G8 transporter (48,49). However, this approach does not address the possibility of post-translational regulation of G5G8 transporter mass observed in the present study.…”

Section: Discussioncontrasting

confidence: 47%

Defects in the Leptin Axis Reduce Abundance of the ABCG5-ABCG8 Sterol Transporter in Liver

Sabeva

Rouse

Graf

2007

ABGG5 (G5) and ABCG8 (G8) are ABC half-transporters that dimerize within the endoplasmic reticulum, traffic to the cell surface, and mediate cholesterol excretion into bile. Mice harboring defects in the leptin axis (db/db and ob/ob) have reduced biliary cholesterol concentrations. Rapid weight loss brought about by administration of leptin or dietary restriction increases biliary cholesterol excretion. We hypothesized that the reduction in biliary cholesterol in mice harboring defects in the leptin axis is associated with a reduction in G5G8 transporters and that levels of the transporter would increase with leptin administration and dietary restriction. We examined mRNA and protein levels for G5 and G8 in db/db and ob/ob mice. In both models G5 and G8 protein levels were reduced. In ob/ob mice, both leptin administration and dietary restriction increased G5 and G8 protein and biliary cholesterol concentrations. Finally, we examined the effects of tauroursodeoxycholate, which has been shown to increase biliary cholesterol excretion and function as a molecular chaperone. Tauroursodeoxycholate increased G5 and G8 protein and biliary cholesterol concentrations in both wild-type and db/db mice. Our results indicate that the mechanism for reduced biliary cholesterol excretion in db/db and ob/ob mice involves reductions in G5 and G8 protein levels and that this may occur at the level of G5G8 heterodimer assembly within the endoplasmic reticulum. ABCG5 (G5) and ABCG8 (G8) play a major role in the elimination of dietary and endogenously synthesized sterols in humans and mice (1-5). G5 and G8 are ATP binding cassette (ABC) 2 half-transporters that dimerize in the endoplasmic reticulum (ER) before trafficking of the functional G5G8 sterol transporter to the apical surface where it promotes the excretion of sterols from hepatocytes and enterocytes (3, 6 -8). In the liver, the G5G8 sterol transporter appears to be the major route for cholesterol excretion into bile. Mice deficient in G5, G8, or both transporters have 80 -90% reductions in biliary cholesterol concentrations (3-5). Conversely, expression of a human transgene in mice results in a 6 -8-fold increase in biliary cholesterol concentration and supersaturation of bile (7).In the absence of dietary or pharmacological perturbations, biliary cholesterol concentrations correlate with Abcg5/Abcg8 genocopy (9). Activation of the liver X receptor (LXR␣, NR1H3) by cholesterol feeding or administration of an agonist increases G5 and G8 mRNA as well as biliary cholesterol concentrations (10). In addition, these effects are absent in mice lacking LXR ␣/␤, suggesting that LXR is the principal regulator of G5 and G8 expression in response to dietary cholesterol (11). Although biliary cholesterol excretion generally correlates with expression levels of G5 and G8 mRNAs, exceptions include increases in biliary cholesterol excretion after treatment with diosgenin or tauroursodeoxycholate (TUDCA) and in liver transplant patients after surgery (12-14). The uncoupling of biliary chole...