Title: Sequences in the Nonconsensus Nucleotide-binding Domain of ABCG5/ABCG8 Required for Sterol Transport
Abstract: 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 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. IntroductionATP-binding cassette (ABC) 3The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain; G5, ABCG5; G8, ABCG8. transporters hydrolyze ATP to transport a wide variety of substrates across membranes (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). 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 (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar). 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 α- and β-phosphates of ATP; the Walker B motif (ϕϕϕϕDE, where ϕ 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 γ-phosphate and closes off the active site (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar). 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 γ-phosphate, and a Q-loop glutamine that plays a role in mediating communication between the NBDs and TMDs (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar).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 (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar, 3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Moody J.E. Thomas P.J. J. Bioenerg. Biomembr. 2005; 37: 475-479Crossref PubMed Scopus (28) Google Scholar, 5Oswald C. Holland I.B. Schmitt L. Naunyn-Schmiedeberg's Arch. Pharmacol. 2006; 372: 385-399Crossref PubMed Scopus (124) Google Scholar, 6Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (929) Google Scholar, 7Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). In several ABC transporters, the two NBDs differ in ATP catalytic activity. These transporters invariably contain one NBD with canonical Walker A, Walker B, and C-loop sequences, whereas the other NBD may contain one or more degenerate motifs. At least 21 ABC transporters have an NBD with non-canonical motifs (8Procko E. Gaudet R. Curr. Opin. Immunol. 2009; 21: 84-91Crossref PubMed Scopus (44) Google Scholar), and functional non-equivalence between the NBDs of some of these transporters has been well documented. For example, in the cystic fibrosis transmembrane conductance regulator (ABCC7), NBD2 binds and rapidly hydrolyzes ATP in a magnesium-dependent manner, whereas NBD1 binds ATP even in the absence of a divalent cation and hydrolyzes ATP very slowly with addition of Mg2+ (9Berger A.L. Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 455-460Crossref PubMed Scopus (76) Google Scholar). The NBDs of transporter associated with antigen processing (ABCB2/ABCB3) also differ in ATPase activity (10Chen M. Abele R. Tampé R. J. Biol. Chem. 2004; 279: 46073-46081Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar): the catalytic activity of NBD2 is essential for driving peptide transport, whereas inactivation of NBD1 results in only decreased transport activity.In some ABC transporters, the two NBDs are encoded by separate transcripts, each of which contributes an NBD and a TMD to the mature complex. The hemitransporters ABCG5 (G5) and ABCG8 (G8) form heterodimers in the endoplasmic reticulum and are transported to the apical membranes of enterocytes and hepatocytes, where they limit the absorption and promote the excretion of neutral sterols (11Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar, 12Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (600) Google Scholar, 13Graf G.A. Li W.P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 14Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Mutations in either G5 or G8 cause the autosomal recessive disease sitosterolemia, which is characterized by accumulation of neutral sterols and premature coronary artery disease (15Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1337) Google Scholar). We showed previously that G5G8 requires only one active ATPase to power sterol transport (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Mutations in the Walker A or B motifs of G5 or in the C-loop of G8 disrupt sterol transport by purified reconstituted G5G8 in vitro and in vivo (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In contrast, mutations in the Walker A and B motifs of G8 and the C-loop of G5 do not interfere with sterol transport.We previously arbitrarily referred to the active NBD of G5G8 as NBD1 and the inactive NBD as NBD2 (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Wang J. Sun F. Zhang D.W. Ma Y. Xu F. Belani J.D. Cohen J.C. Hobbs H.H. Xie X.S. J. Biol. Chem. 2006; 281: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Wang J. Zhang D.W. Lei Y. Xu F. Cohen J.C. Hobbs H.H. Xie X.S. Biochemistry. 2008; 47: 5194-5204Crossref PubMed Scopus (35) Google Scholar). 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 (9Berger A.L. Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 455-460Crossref PubMed Scopus (76) Google Scholar). To investigate the determinants and the consequences of functional asymmetry in the G5G8 NBDs, we used an in vivo sterol transport assay. The assay takes advantage of the fact that mice lacking G5 or G8 cannot secrete sterols into bile (11Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar) and that biliary sterol transport can be restored in these animals by using recombinant adenoviruses to coexpress G5 and G8 in the liver.DISCUSSIONIn this work, we examined the role of NBD1 of G5G8 in sterol transport. 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 (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Wang J. Sun F. Zhang D.W. Ma Y. Xu F. Belani J.D. Cohen J.C. Hobbs H.H. Xie X.S. J. Biol. Chem. 2006; 281: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). A major finding of this study is that NBD1, although functionally degenerate, is not dispensable for sterol transport. Although disruption of individual motifs and canonical residues of NBD1 had no appreciable effect on sterol transport in vivo, mutation of the Walker A motif and the C-loop of NBD1 together markedly reduced cholesterol transport while preserving efflux of non-cholesterol sterols. These data are consistent with a model in which NBD1 plays a structural rather than a catalytic role in G5G8-mediated sterol transport.The mechanism by which alterations in NBD1 affect substrate specificity is not known. A similar alteration in substrate selectivity was seen when the highly conserved lysine in the Walker A motif of NBD2 was replaced with arginine (K93R) (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Thus, it is possible that the mutations in NBD1 alter substrate specificity by altering the rate of ATP hydrolysis in NBD2. The transport of cholesterol by G5G8 may be more energetically costly than that of the plant sterols and thus be more susceptible to mutations that reduce the efficiency of ATP hydrolysis.Alternatively, mutations in NBD1 and NBD2 of G5G8 may alter the substrate specificity of the transporter by changing the kinetics of specific steps of the catalytic cycle, rather than the overall rate of ATP hydrolysis. Ernst et al. (22Ernst R. Kueppers P. Klein C.M. Schwarzmueller T. Kuchler K. Schmitt L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5069-5074Crossref PubMed Scopus (112) Google Scholar) showed that mutation of the switch-loop histidine (H1068A) in the yeast multidrug ABC transporter Pdr5 does not influence ATP hydrolysis but abolishes the transport of rhodamine while retaining the ability to transport other substrates. Those authors proposed that by specifically altering nucleotide binding, hydrolysis, or release, the H1068A mutation alters the equilibration time of transport substrates with the inward-facing drug-binding site. For substrates with different on- or off-rates (Kon or Koff), changes in equilibration time could lead to differential changes in transport efficiency, thereby altering substrate specificity. Thus, changes in the Walker A motif of NBD2 or in Walker A and the C-loop of NBD1 of G5G8 could alter substrate specificity by affecting the ATP catalytic cycle, rather than by changing the substrate-binding architecture of the transporter.A third possibility is that mutations in the NBDs could alter substrate specificity by altering the interactions between the NBDs and TMDs. Beaudet et al. (23Beaudet L. Urbatsch I.L. Gros P. Biochemistry. 1998; 37: 9073-9082Crossref PubMed Scopus (45) Google Scholar) found that selected mutations around the Walker B motif of NBD1 in P-glycoprotein alter substrate specificity of the transporter. The mutations did not influence substrate binding but selectively altered substrate-induced ATPase activity. Accordingly, the authors proposed that the residues involved may mediate communication between the NBDs and TMDs. Although structural studies suggest that the Walker A lysine is not exposed to the TMDs during the catalytic cycle, we cannot exclude the possibility that mutations in NBD1 and NBD2 that alter substrate specificity act by altering a signaling relay between the TMDs and NBDs.Degeneracy of NBD1 is not required for G5G8 function, as substitution of the degenerate motifs with the canonical sequences neither impaired nor augmented G5G8-mediated sterol transport. This finding indicates that the predicted loss of ATPase activity that results from the degenerate motifs in NBD1 has little direct impact on G5G8 function. If NBD1 is not required to catalyze ATP hydrolysis, and if the degenerate motifs do not serve a specific purpose, what is the role of NBD1 in G5G8 function? In other asymmetric ABC transporters such as the cystic fibrosis transmembrane conductance regulator, the degenerate NBD retains the ability to bind ATP (21Aleksandrov L. Aleksandrov A.A. Chang X.B. Riordan J.R. J. Biol. Chem. 2002; 277: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). To test the hypothesis that ATP binding to NBD1 of G5G8 is required for transporter function, we designed a series of mutations in the Walker A motif of G8 that would be expected to disrupt ATP binding. None of these mutations interfered with sterol transport activity (Fig. 6). However, we were unable to confirm that the mutations completely disrupted ATP binding because all of the mutant proteins bound 8-azido-[α-32P]ATP in the presence of an inhibitor, beryllium fluoride (data not shown). It is possible that the ATP that is covalently linked to G8 in these experiments bound to the C-loop of NBD2 (which is contributed by G8) rather than to NBD1. Alternatively, the mutant NBD1 sites in these proteins may bind ATP with sufficient affinity to affect sterol transport even when the sequence of the Walker A motif is disrupted.To further investigate possible determinants of ATP binding to NBD1, we mutated the aromatic residues corresponding to the A-loops in NBD1 and NBD2. These mutations did not alter sterol transport in vivo, suggesting that the A-loop residues do not play a critical role in ATP binding to G5G8. It remains possible that other amino acids in G5G8 coordinate binding to the nucleotide ring, as they do in members of the ABCD family, where isoleucine or leucine residues serve this function (20Ambudkar S.V. Kim I.W. Xia D. Sauna Z.E. FEBS Lett. 2006; 580: 1049-1055Crossref PubMed Scopus (130) Google Scholar).In contrast to NBD1, preservation of the ATPase activity of NBD2 of G5G8 is both necessary and sufficient for sterol transport. Mutating key residues predicted to disrupt ATP hydrolysis by this domain abolishes G5G8 function. The observation that the switch-loop histidine is essential for sterol transport indicates a critical role for this residue in ATP hydrolysis. This finding is consistent with the model proposed by Schmitt and co-workers (24Zaitseva J. Jenewein S. Jumpertz T. Holland I.B. Schmitt L. EMBO J. 2005; 24: 1901-1910Crossref PubMed Scopus (283) Google Scholar) in which the glutamate of Walker B and the switch-loop histidine form a catalytic dyad. Interestingly, neither the D-loop aspartate nor the Q-loop glutamine of NBD2 is required for G5G8-mediated sterol transport, suggesting that neither residue contributes significantly to ATP hydrolysis by this transporter.The location of ATPase activity in NBD2 is also critical. Transposing the consensus sequences of the two NBDs preserved expression and trafficking of the transporter but eliminated all sterol transport. Among 19 full transporters with degenerate ATPase motifs, the non-canonical sequences are invariably in NBD1 (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar). In summary, these findings suggest that ATPase activity in NBD2 is essential for substrate transport by ABC transporters. IntroductionATP-binding cassette (ABC) 3The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain; G5, ABCG5; G8, ABCG8. transporters hydrolyze ATP to transport a wide variety of substrates across membranes (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). 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 (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar). 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 α- and β-phosphates of ATP; the Walker B motif (ϕϕϕϕDE, where ϕ 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 γ-phosphate and closes off the active site (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar). 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 γ-phosphate, and a Q-loop glutamine that plays a role in mediating communication between the NBDs and TMDs (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar).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 (2Procko E. O'Mara M.L. Bennett W.F. Tieleman D.P. Gaudet R. FASEB J. 2009; 23: 1287-1302Crossref PubMed Scopus (118) Google Scholar, 3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Moody J.E. Thomas P.J. J. Bioenerg. Biomembr. 2005; 37: 475-479Crossref PubMed Scopus (28) Google Scholar, 5Oswald C. Holland I.B. Schmitt L. Naunyn-Schmiedeberg's Arch. Pharmacol. 2006; 372: 385-399Crossref PubMed Scopus (124) Google Scholar, 6Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (929) Google Scholar, 7Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). In several ABC transporters, the two NBDs differ in ATP catalytic activity. These transporters invariably contain one NBD with canonical Walker A, Walker B, and C-loop sequences, whereas the other NBD may contain one or more degenerate motifs. At least 21 ABC transporters have an NBD with non-canonical motifs (8Procko E. Gaudet R. Curr. Opin. Immunol. 2009; 21: 84-91Crossref PubMed Scopus (44) Google Scholar), and functional non-equivalence between the NBDs of some of these transporters has been well documented. For example, in the cystic fibrosis transmembrane conductance regulator (ABCC7), NBD2 binds and rapidly hydrolyzes ATP in a magnesium-dependent manner, whereas NBD1 binds ATP even in the absence of a divalent cation and hydrolyzes ATP very slowly with addition of Mg2+ (9Berger A.L. Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 455-460Crossref PubMed Scopus (76) Google Scholar). The NBDs of transporter associated with antigen processing (ABCB2/ABCB3) also differ in ATPase activity (10Chen M. Abele R. Tampé R. J. Biol. Chem. 2004; 279: 46073-46081Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar): the catalytic activity of NBD2 is essential for driving peptide transport, whereas inactivation of NBD1 results in only decreased transport activity.In some ABC transporters, the two NBDs are encoded by separate transcripts, each of which contributes an NBD and a TMD to the mature complex. The hemitransporters ABCG5 (G5) and ABCG8 (G8) form heterodimers in the endoplasmic reticulum and are transported to the apical membranes of enterocytes and hepatocytes, where they limit the absorption and promote the excretion of neutral sterols (11Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar, 12Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (600) Google Scholar, 13Graf G.A. Li W.P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 14Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Mutations in either G5 or G8 cause the autosomal recessive disease sitosterolemia, which is characterized by accumulation of neutral sterols and premature coronary artery disease (15Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1337) Google Scholar). We showed previously that G5G8 requires only one active ATPase to power sterol transport (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Mutations in the Walker A or B motifs of G5 or in the C-loop of G8 disrupt sterol transport by purified reconstituted G5G8 in vitro and in vivo (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In contrast, mutations in the Walker A and B motifs of G8 and the C-loop of G5 do not interfere with sterol transport.We previously arbitrarily referred to the active NBD of G5G8 as NBD1 and the inactive NBD as NBD2 (3Zhang D.W. Graf G.A. Gerard R.D. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2006; 281: 4507-4516Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Wang J. Sun F. Zhang D.W. Ma Y. Xu F. Belani J.D. Cohen J.C. Hobbs H.H. Xie X.S. J. Biol. Chem. 2006; 281: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Wang J. Zhang D.W. Lei Y. Xu F. Cohen J.C. Hobbs H.H. Xie X.S. Biochemistry. 2008; 47: 5194-5204Crossref PubMed Scopus (35) Google Scholar). 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 (9Berger A.L. Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 455-460Crossref PubMed Scopus (76) Google Scholar). To investigate the determinants and the consequences of functional asymmetry in the G5G8 NBDs, we used an in vivo sterol transport assay. The assay takes advantage of the fact that mice lacking G5 or G8 cannot secrete sterols into bile (11Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar) and that biliary sterol transport can be restored in these animals by using recombinant adenoviruses to coexpress G5 and G8 in the liver.