ABCMdb

PMID: 16107343

Henriksen U, Fog JU, Litman T, Gether U
Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2.
J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17., 2005-11-04 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Login to comment 4 ABCG2 p.Cys603Ala Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Login to comment 5 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. Login to comment 6 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Login to comment 7 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/ C608A), the transporter displayed impaired plasma membrane expression and function. Login to comment 8 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. Login to comment 25 ABCG2 p.Lys86Met However, our previous data also shows that dimerization of the transporter is not dependent on functional nucleotide binding domains, as an inactivating mutation (K86M) in the Walker A motif did not alter the ability of ABCG2 to form dimers (24). Login to comment 32 ABCG2 p.Arg482Gly MATERIALS AND METHODS Plasmids, Drugs, and Antibodies-pcDNA3.1(-)MXR (R482G) and fumitremorgin C (FTC) was kindly provided by Dr. Susan Bates, NCI, National Institutes of Health. Login to comment 104 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala The resulting constructs (C592A, C603A, and C608A) were stably expressed in HEK293 cells using the bicistronic vector pCIN4 (28), and total cell lysates were analyzed by Western blotting in the presence of increasing concentrations of DTT (Fig. 3). Login to comment 107 ABCG2 p.Cys603Ala Interestingly, the disulfide bridge-linked dimer was completely absent in the C603A mutant (Fig. 3B). Login to comment 108 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala In C592A and C608A, the disulfide-linked dimer was still present, although only scarcely in Cys-608 (Fig. 3, A and C). Login to comment 109 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala We observed also, however, a significant amount of monomer in the unreduced samples of C592A and C608A as compared with wt (Fig. 3, A and C). Login to comment 113 ABCG2 p.Cys603Ala Subsequent immunocytochemistry analysis of the single mutants was in agreement with the Western blot analysis and supported that the wt and C603A mutant were expressed almost exclusively in the plasma membrane (Fig. 3, B and D). Login to comment 114 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala In comparison, C592A and C608A exhibited less apparent plasma membrane staining and substantially more intracellular staining than in wt and C603A (Fig. 3, A and C). Login to comment 118 ABCG2 p.Cys603Ala Only in C603A, the resistance FIGURE 3. Login to comment 120 ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala C608A (A), C603A (B),C592A(C),andwt(D)areshown.Immunostainings of the corresponding cells are shown directly below. Login to comment 122 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala The samples were analyzed by confocal microscopy using a Zeiss LSM510. Cysteine-linked Dimerization of ABCG2 36928 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280•NUMBER 44•NOVEMBER , to mitoxantrone was directly comparable with wt, whereas C592A and C608A showed significant decrease in resistance of ϳ50 and 70%, respectively (Fig. 4B). Login to comment 125 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala The analysis showed that, although C603A tended to have a higher total expression, there were no significant changes in the expression of C592A and C608A. Login to comment 126 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala It is, however, important to correlate this with the immunostainings; i.e. both C592A and C608A displayed less apparent plasma membrane staining and increased intracellular staining, possibly accounting for the reduced activity (Fig. 4B). Login to comment 128 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala The impaired targeting of C592A and C608A led us to hypothesize that these two residues could form a structurally important intramolecular disulfide bridge. Login to comment 129 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala To further explore this hypothesis, we mutated two cysteines at a time, resulting in C592A/C603A, C592A/C608A, and C603A/C608A, each of which contained one remaining extracellular cysteine. Login to comment 131 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala In C592A/C608A, which contained only Cys-603, we observed, in agreement with our hypothesis, efficient dimerization (Fig. 5D). Login to comment 132 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala The expression of C592A/C608A was also high and, in fact, increased as compared with the wt (Fig. 6C). Login to comment 133 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala In contrast, protein expression was very low in the construct containing only Cys-592 (C603A/ C608A) (Figs. 5C and 6C) and slightly lower than wt for C592A/C603A, in which only Cys-608 is present (Figs. 5A and 6C). Login to comment 134 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Notably, we also detected dimer formation in C603A/C608A and C592A/C603A. Login to comment 136 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala A, comparison of cell survival between non-transfected HEK293 cells (E) and HEK293 cells transfected with ABCG2-wt (●), C592A (‚), C603A (ƒ), and C608A (Ⅺ). Login to comment 144 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala ABCG2-C592A/C608A containing only Cys-603 dimerizes efficiently and is highly expressed. Login to comment 145 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala Shown are C592A/C603A (A), wt (B), C603A/C608A (C), and C592A/ C608A (D). Login to comment 152 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala Staining of the transfected HEK293 cells showed that, although C592A/C608A (Fig. 5D) mostly resembled the membrane-localized expression pattern of wt (Fig. 5B), C592A/C603A displayed largely intracellular staining (Fig. 5A), and C603A/C608A was hardly visible (Fig. 5C). Login to comment 157 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala For both C603A/C608A and C592A/C603A, we observed a marked decrease in resistance to mitoxantrone and a concomitant decrease in expression (Fig. 6, B and C). Login to comment 158 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala For C592A/C608A, we observed, nonetheless, an increase in expression (Fig. 6C). Login to comment 160 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala We also substituted all three extracellular cysteines in ABCG2 simultaneously (C592A/C603A/C608A). Login to comment 162 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Specifically, we were almost unable to detect any expressed protein in the immunostainings (Fig. 7B) To further explore the function of the hypothesized disulfide bridges, we performed efflux experiments on the mutants containing either both Cys-592 and Cys-608 (C603A), predicted to form an intramolecular disulfide bridge, or Cys-603 only (C592A/C608A), predicted to form an intermolecular disulfide bridge (Fig. 9). Login to comment 164 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala C603A displayed efflux similar to wt, whereas efflux in C592A-C608A was slightly decreased (Fig. 9). Login to comment 165 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala The pattern was, however, different when analyzing another substrate for ABCG2, BODIPY-prazosin; i.e. we observed BODIPY-prazosin efflux similar to the wt in C603A, whereas in C592A-C608A we could not detect any evidence for BODIPY-prazosin efflux (Fig. 9). Login to comment 168 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Without prior TCEP reduction, we observed no labeling consistent with no cysteines on the extracellular face of the transporter available for biotinylation (Fig. 10); however, upon TCEP treatment, we found clear biotin labeling of both C603A and C592A/C608A. Login to comment 172 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala A, comparison of cell survival between empty HEK293 (E) and ABCG2-wt (●), C592A/C603A (‚), C592A/C608A (ƒ), and C603A/C608A (Ⅺ). Login to comment 181 ABCG2 p.Cys603Ala Of all mutants tested, only C603A showed clear staining with anti-ABCG2 (5D3) (Fig. 11); all other mutants showed no staining (Fig. 11). Login to comment 196 ABCG2 p.Cys603Ala The unconstrained plasma membrane targeting of C603A also indicates that we have not disrupted dimerization in the native membrane, because evidence has been obtained in the homologous FIGURE 7. Login to comment 202 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala The transmitted light image of the C592A/C603A/C608A (3cys) sample is also shown. Login to comment 206 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Empty HEK293 (●), ABCG2-wt (Œ), and ABCG2-C592A/C603A/C608A (3cys) (f) are shown. Login to comment 217 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala However, simultaneous mutation of both cysteines (C592A/C608A) restored plasma membrane targeting, and the expression even tended to be higher than that observed for the wt (Fig. 5). Login to comment 219 ABCG2 p.Cys603Ala In addition, biochemical evidence for the existence of this disulfide bridge was obtained from the biotinylation experiment showing biotinylation in C603A only with prior TCEP reduction (Fig. 10). Login to comment 221 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Cys-592 and Cys-608 might nonetheless form intermolecular disulfide bridges when they are "alone" as indicated from the results with the double mutations; i.e. both in C603A/C608A (where Cys-592 is alone) and in C592A/C603A (where Cys-608 is alone), we observed some disulfide bridge-linked dimers despite the fact that Cys603 is mutated. Login to comment 223 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Efflux of BODIPY-prazosin and mitoxantrone is not affected by removal of the intermolecular disulfide bridge (C603A), whereas removal of the intramolecular disulfide bridge affects BODIPY-prazosin efflux (C592A/C608A). Login to comment 231 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Cells stably expressing wt, C603A, or C592A/C608A ABCG2 were exposed to a cysteine-reactive biotinylation agent after incubation with (ϩ) or without (-) the reducing agent TCEP. Login to comment 236 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala In this context, it is also interesting to pay attention to the finding that, in the single mutants (C592A and C608A), we observed a significant amount of monomer in the unreduced samples (Fig. 3, A and C). Login to comment 241 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Mutating all three extracellular cysteines in ABCG2 (C592A/C603A/ C608A) at the same time had detrimental effects on the transporter. Login to comment 243 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala This supports the conclusion that, although we can disrupt the putative intermolecular symmetrical disulfide bridge involving Cys-603 (C603A mutant) and although we can remove the putative intramolecular disulfide bridge between Cys-592 and Cys-608 (C592A/C608A mutant) without any major impact on expression of the transporter, it is not possible to remove both of them simultaneously. Login to comment 246 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala In C592A/C608A, we see no efflux of the substrate BODIPY-prazosin, although efflux of mitoxantrone is preserved. Login to comment