RÉSUMÉ
ABCA1 plays a key role in cellular cholesterol and phospholipid traffic. To explore the biochemical properties of this membrane protein we applied a Baculovirus-insect cell expression system. We found that human ABCA1 in isolated membranes showed a specific, Mg(2+)-dependent ATP binding but had no measurable ATPase activity. Nevertheless, conformational changes in ABCA1 could be demonstrated by nucleotide occlusion, even without arresting the catalytic cycle by phosphate-mimicking anions. Addition of potential lipid substrates or lipid acceptors (apolipoprotein A-I) did not modify the ATPase activity or nucleotide occlusion by ABCA1. Our data indicate that ATP hydrolysis by ABCA1 occurs at a very low rate, suggesting that ABCA1 may not function as an effective active transporter as previously assumed. In the light of the observed conformational changes we propose a regulatory function for human ABCA1.
Sujet(s)
Transporteurs ABC/physiologie , Adenosine triphosphatases/métabolisme , Membre-1 de la sous-famille A des transporteurs à cassette liant l'ATP , Transporteurs ABC/génétique , Animaux , Apolipoprotéine A-I/pharmacologie , Baculoviridae/génétique , Transport biologique actif , Vecteurs génétiques , Humains , Membranes intracellulaires/métabolisme , Métabolisme lipidique , Spodoptera/génétique , TransfectionRÉSUMÉ
ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A. Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity. The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer. We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents.
Sujet(s)
Transporteurs ABC/physiologie , Protéines tumorales , Membre-2 de la sous-famille G des transporteurs à cassette liant l'ATP , Transporteurs ABC/génétique , Adenosine triphosphatases/métabolisme , Animaux , Tumeurs du sein/anatomopathologie , Membrane cellulaire/enzymologie , Clonage moléculaire , Multirésistance aux médicaments , Résistance aux médicaments antinéoplasiques , Humains , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Spodoptera , Cellules cancéreuses en cultureRÉSUMÉ
The human multidrug resistance protein (MDR1) (P-glycoprotein), a member of the ATP-binding cassette (ABC) family, causes multidrug resistance by an active transport mechanism, which keeps the intracellular level of hydrophobic compounds below a cell-killing threshold. Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D/G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes. These mutations caused a loss of measurable ATPase activity but still allowed ATP binding and the formation of a transition-state intermediate (nucleotide trapping). In contrast with the wild-type protein, in which substrate drugs accelerate nucleotide trapping, in the ABC signature mutants nucleotide trapping was inhibited by MDR1-substrate drugs, suggesting a miscommunication between the drug-binding site(s) and the catalytic domains. Equivalent mutations of the two catalytic sites resulted in a similar effect, indicating the functional equivalence of the two sites. On the basis of these results and recent structural information on an ABC-ABC dimer [Hopfner, Karcher, Shin, Craig, Arthur, Carney and Tainer (2000) Cell 101, 789-800], we propose a key role of these glycine residues in the interdomain communication regulating drug-induced ATP hydrolysis.
Sujet(s)
Glycoprotéine P/génétique , Glycoprotéine P/métabolisme , Adénosine triphosphate/métabolisme , Multirésistance aux médicaments/génétique , Glycine/génétique , Régulation allostérique , Transport biologique actif , Variation génétique , Humains , Hydrolyse , Mutation , Protéines recombinantes/métabolismeRÉSUMÉ
In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Both mutations have been demonstrated to cause a loss of drug transport, drug-stimulated ATPase, and vanadate-dependent nucleotide trapping activity. Here we show that these mutants still allow transition state formation (nucleotide trapping) when fluoro-aluminate or beryllium fluoride is used as a complex-stabilizing anion. Drug stimulation of nucleotide trapping was found to be preserved in both mutants. Limited trypsin digestion revealed that whenever MDR1-nucleotide trapping occurred, both ABC domains were involved in the formation of the catalytic intermediates. Our results show that details of the MDR1-ATPase cycle can be studied even in ATPase-negative mutants. These data also demonstrate that the conformational alteration caused by a mutation in one of the ABC domains is propagated to the other, nonmutated domain, indicating a tight coupling between the functioning of the two ABC domains.