ABCMdb

PMID: 9829963

Loo TW, Clarke DM
Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein.
J Biol Chem. 1998 Dec 4;273(49):32373-6., 1998-12-04 [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala A glycosylation-deficient mutant was made by mutating the three consensus glycosylation sites (N91A,N94A,N99A). Login to comment 64 ABCB1 p.Gly341Cys For some mutants such as G341C, no full-length protein could be detected. Login to comment 67 ABCB1 p.Gly341Cys To inhibit or retard the degradation of mutant G341C, synthesis was carried out in the presence of proteasome inhibitors such as MG-132 or lactacystin, because the major cellular degradation pathway is mediated by proteasomes (17). Login to comment 68 ABCB1 p.Gly341Cys Fig. 1 (lanes 8 and 9) shows that proteasomes are probably not involved because the 130-kDa protein was still the major product when mutant G341C was synthesized in the presence of proteasome inhibitors. Login to comment 69 ABCB1 p.Gly341Cys In contrast to mutant G341C, the proteasome inhibitors had a dramatic effect on synthesis of the Cys-less parent. Login to comment 71 ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala The 130-kDa product was also observed when the glycosylation-deficient (N91A,N94A,N99A) mutant was expressed in the presence of proteasome inhibitors (Fig. 1, lanes 5 and 6). Login to comment 73 ABCB1 p.Gly341Cys Therefore, the presence of a degradation product of 130 kDa in the Cys-less parent P-gp and in mutant G341C suggested that the protein had been cleaved at the NH2-terminal end. Login to comment 78 ABCB1 p.Arg113Cys As shown in Fig. 2B, the 130-kDa product of mutant R113C was not detected, whereas the amount of the core-glycosylated 150-kDa protein was increased. Login to comment 79 ABCB1 p.Tyr114Cys Mutant Y114C showed reduced amounts of the 130-kDa product and increased amounts of core-glycosylated product. Login to comment 93 ABCB1 p.Gly341Cys ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala HEK 293 cells were transfected with Cys-less (C-less), mutant G341C (in Cys-less background), or glycosylation-deficient (N91A,N94A,N99A; -Glycos.) Login to comment 104 ABCB1 p.Arg113Met ABCB1 p.Arg113Glu ABCB1 p.Arg113Lys We found that mutation of Arg113 to Lys, Met, Glu, Ala, or Cys all blocked cleavage of P-gp (Fig. 3B, lanes 2-7). Login to comment 107 ABCB1 p.Tyr114Cys ABCB1 p.Tyr114Glu ABCB1 p.Tyr114Met ABCB1 p.Tyr114Lys Mutation of Tyr114 to Cys, Lys, Met, or Glu resulted in reduced amounts of 130-kDa product in the presence of MG-132. Login to comment 108 ABCB1 p.Tyr114Ala In contrast, no 130-kDa product was observed in mutant Y114A (Fig. 3B). Login to comment 114 ABCB1 p.Gly268Val ABCB1 p.Arg113Ala Accordingly, cells expressing a misprocessed mutant (G268V) with or without an additional R113A mutation were radiolabeled with [35 S]methionine and [35 S]cystine to monitor the fate of the mutant. Login to comment 115 ABCB1 p.Gly268Val Mutant G268V is a temperature-insensitive processing mutant (8) that yields mature enzyme (170 kDa) only when synthesized in the presence of drug substrates (7). Login to comment 116 ABCB1 p.Arg113Ala Fig. 4 shows that there was no detectable difference between the maturation of the core-glycosylated wild type enzyme and the (wild type ϩ R113A) mutant. Login to comment 117 ABCB1 p.Gly269Val ABCB1 p.Gly268Val ABCB1 p.Gly268Val ABCB1 p.Arg113Ala By contrast, the core-glycosylated G268V protein failed to mature and was almost undetectable by 24 h. In the double mutant (G268V ϩ R113A), however, there was a 5-10-fold increase in the amount of P-gp at 0 h, but the mutant still failed to mature and was degraded by 24 h. A similar pattern was observed for the processing mutant G269V (data not shown). Login to comment 118 ABCB1 p.Gly268Val ABCB1 p.Gly341Cys Mutant G341C appeared to have a greater folding defect than the G268V misprocessed mutant. Login to comment 122 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys The R113A mutation was then introduced to block cleavage of the G341C mutant. Login to comment 123 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys As shown in Fig. 5A (lane 3), the introduction of an R113A mutation significantly affected the synthesis of mutant G341C. Login to comment 126 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys To determine whether the mature mutant protein was functional, the histidine-tagged mutant (G341C ϩ R113A) was expressed in HEK 293 cells and purified by nickel-chelate chromatography. Login to comment 129 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys Mutant (G341C ϩ R113A) exhibited verapamiland vinblastine-stimulated ATPase activities that were similar to the parent enzyme. Login to comment 130 ABCB1 p.Gly341Cys These results show that blockage of the Arg113 cleavage site completely rescued the defects associated with the G341C mutation. Login to comment 131 ABCB1 p.Gly341Cys We could not determine whether the 130-kDa product of mutant G341C was active because the histidine-tagged protein was not retained by the nickel column, a property also exhibited by the core-glycosylated intermediates of other P-gp processing mutants (10). Login to comment 132 ABCB1 p.Gly341Cys The inability of the histidine-tagged 130-kDa product of mutant G341C to bind the nickel column was not due to loss of the histidine tag. Login to comment 134 ABCB1 p.Gly268Val ABCB1 p.Gly268Val ABCB1 p.Arg113Ala ABCB1 p.Arg113Ala FIG. 4. Effect of R113A mutation on maturation of wild type and mutant G268V P-gp. HEK 293 cells expressing wild type or mutant G268V P-gps-A52 and containing Arg113 or R113A were pulse-labeled for 20 min at 37 °C and then chased with fresh media for the various intervals (hr). Login to comment 137 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys FIG. 5. Effect of R113A mutation on maturation and drug-stimulated ATPase activity of mutant G341C. Login to comment 138 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys ABCB1 p.Gly341Cys A, HEK 293 cells expressing P-gp-A52 mutants G341C or G341C ϩ R113A in Cys-less background were grown with (ϩ) or without (-) 10 ␮M cyclosporin A for 24 h. Equivalent amounts of whole cell extracts were subjected to immunoblot analysis with monoclonal antibody A52. Login to comment 139 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys B, purified histidine-tagged Cys-less or mutant G341C ϩ R113A (in Cys-less background P-gp) were added to lipid and assayed for ATPase with or without verapamil (1 mM, shaded bars) or vinblastine (0.05 mM, unshaded bars). Login to comment 146 ABCB1 p.Gly341Cys This effect was enhanced in mutant G341C, such that the 130-kDa protein was the only product detected. Login to comment 147 ABCB1 p.Gly341Cys Remarkably, the effects of the G341C mutation could be overcome by mutating out the hypersensitive protease cleavage site. Login to comment 148 ABCB1 p.Arg113Ala ABCB1 p.Gly341Cys Apparently, the G341C mutation does not cause complete misfolding of the protein because introduction of an additional R113A mutation to prevent degradation converts the mutant to a functional enzyme with characteristics indistinguishable from parent enzyme (Fig. 5B). Login to comment 158 ABCB1 p.Gly341Cys By inhibiting digestion at Arg113 , it was possible to increase expression of P-gp mutants such as G341C. Login to comment