ABCMdb

PMID: 9614062

Loo TW, Clarke DM
Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions.
J Biol Chem. 1998 Jun 12;273(24):14671-4., 1998-06-12 [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCB1 p.Gly427Cys ABCB1 p.Glu707Ala ABCB1 p.Ala123Leu ABCB1 p.Glu243Ala ABCB1 p.Tyr853Cys ABCB1 p.Pro1194Ala Misprocessing mutations are located throughout the molecule; these include the transmembrane domains (e.g. A123L), intracellular (e.g. E243A) and extracellular (e.g. Y853C) loops, the linker region (e.g. E707A), and both nucleotide-binding domains (e.g. G427C, P1194A). Login to comment 52 ABCB1 p.Gly268Val When mutant G268V was expressed in HEK 293 cells (no drug, lane 1), the major product was a 150-kDa core-glycosylated protein that was sensitive to digestion with endoglycosidase H (data not shown). Login to comment 54 ABCB1 p.Gly268Val We attempted to purify the core-glycosylated intermediate of mutant G268V to determine whether it was still active. Login to comment 63 ABCB1 p.Gly268Val As shown in Fig. 1A (no drug), the 150-kDa protein of mutant G268V was quite sensitive to trypsin. Login to comment 74 ABCB1 p.Gly268Val To test this possibility, we expressed mutant G268V in the presence of a proteasome inhibitor (MG-132). Login to comment 76 ABCB1 p.Gly268Val As shown in Fig. 1C (lanes 3 and 4), incubation of mutant G268V for 24 h in the presence of MG-132 resulted in accumulation of the core-glycosylated intermediate and a 130-kDa digestion product. Login to comment 78 ABCB1 p.Gly268Val Increased accumulation of the core-glycosylated intermediate and the 130-kDa digestion product was also seen with the FIG. 1. Protease sensitivity of mutant G268V and wild-type P-glycoproteins. Login to comment 79 ABCB1 p.Gly268Val Membranes prepared from HEK 293 cells expressing mutant G268V (A) or wild-type (B) P-glycoproteins-A52 grown without (no drug) or with (ϩ drug) 10 ␮M CsA were treated with TPCK-trypsin followed by immunoblot analysis. Login to comment 81 ABCB1 p.Gly268Val C, HEK 293 cells transfected with wild-type or G268V cDNAs were incubated for 24 h in media containing 5 ␮M MG132 (ϩ) or no inhibitor (-), and equivalent amounts of whole cell extracts were immunoblotted with monoclonal antibody A52. Login to comment 89 ABCB1 p.Gly268Val We first introduced a processing mutation (G268V) into N-Half-His. Login to comment 90 ABCB1 p.Gly268Val ABCB1 p.Gly268Val Unlike the histidine-tagged full-length G268V P-glycoprotein, the mutant (G268V) N-Half-His could be recovered by nickel-chelate chromatography, even when grown with no CsA (Fig. 2A). Login to comment 91 ABCB1 p.Gly268Val Wild-type or G268V N-Half-His molecules were then coexpressed with the C-Half-A52 molecule. Login to comment 93 ABCB1 p.Gly268Val As shown in Fig. 2B, C-Half-A52 was not retained on the column when expressed alone or when coexpressed with mutant G268V N-Half- His in the absence of CsA. Login to comment 94 ABCB1 p.Gly268Val In contrast, CsA promoted interaction between C-Half-A52 and mutant G268V N-Half-His, such that most of the C-Half-A52 was now recovered by nickel-chelate chromatography. Login to comment 97 ABCB1 p.Ala841Leu We then tested the effect of a misprocessing mutation located in the C-half of P-glycoprotein (A841L), on the ability of the two halves to associate. Login to comment 99 ABCB1 p.Ala841Leu Wild-type N-Half-A52 was coexpressed in HEK 293 cells with either wild-type C-Half-His or mutant (A841L) C-Half-His, in the presence or absence of CsA. Login to comment 100 ABCB1 p.Ala841Leu Fig. 3A shows that CsA had a dramatic effect on the association of A841L C-Half-His and wild-type N-Half-A52. Login to comment 111 ABCB1 p.Gly268Val A, membranes from HEK 293 cells expressing histidine-tagged wild-type or G268V N-half molecules were treated for 24 h with (ϩ) or without (-) 10 ␮M CsA, solubilized with 1% (w/v) n-dodecyl-beta-D-maltoside, and the supernatants were subjected to nickel-chelate chromatography. Login to comment 113 ABCB1 p.Gly268Val B, detergent-solubilized HEK 293 membranes expressing only C-Half-A52, C-Half-A52, and wild-type N-Half-His, or both C-Half-A52 and G268V N-Half-His grown with (ϩ) or without (-) 10 ␮M CsA were subjected to nickel-chelate chromatography. Login to comment 117 ABCB1 p.Ala841Leu A, wild-type N-Half-A52 was coexpressed with wild-type C-Half-His or with mutant (A841L) C-Half-His in the presence (ϩ) or absence (-) of 10 ␮M CsA. Login to comment 120 ABCB1 p.Gly268Val B, wild-type or mutant (G268V) N-Half-A52 was coexpressed with wild-type C-Half-His in the presence of 10 ␮M CsA. Login to comment