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Abstract
ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) belongs to the ABC transporter superfamily and has been implicated in multidrug resistance of cancers. Although the structure and function of ABCG2 have been extensively studied, little is known about its biogenesis and the regulation thereof. In this study, using mutagenesis and several biochemical analyses, we show that the positive charges in the vicinity of the RKR motif downstream of the ABC signature drive trafficking of nascent ABCG2 out of the endoplasmic reticulum (ER) onto plasma membranes. Substitutions of and naturally occurring single-nucleotide polymorphisms within these positively charged residues disabled the trafficking of ABCG2 out of the ER. A representative ABCG2 variant in which the RKR motif had been altered underwent increased ER stress-associated degradation. We also found that unlike WT ABCG2, genetic ABCG2 RKR variants have disrupted normal maturation and do not reduce accumulation of the anticancer drug mitoxantrone and no longer confer resistance to the drug. We conclude that the positive charges downstream of the ABC signature motif critically regulate ABCG2 trafficking and maturation. We propose that single-nucleotide polymorphisms of these residues reduce ABCG2 expression via ER stress-associated degradation pathway and may contribute to reduced cancer drug resistance, improving the success of cancer chemotherapy.
Highlights
Subfamily G member 2 (ABCG2) belongs to ATPbinding cassette (ABC) transporter superfamily and has been implicated in multidrug resistance of cancers
Because loss of positive charges downstream of the C-motif inhibits ABCG2 trafficking from endoplasmic reticulum (ER) to plasma membranes and induces ABCG2 degradation in proteasomes as shown above, we hypothesized that these mutations might increase accumulation of the mutant ABCG2 in ER by inhibiting its trafficking, resulting in ER stress and degradation of these mutant ABCG2 via ER stressassociated degradation (ERAD) pathway
In this study, we found that the positive charges immediate downstream of the C-motif plays an important role in ABCG2 biogenesis
Summary
Sequence analyses using an online tool (https://scansite4.mit.edu) showed two potential AKT phosphorylation sites, T194 and S195, downstream of the C-motif in ABCG2 (Figure 1A) To investigate if these sites are important, we mutated T194 and S195 to create non-phosphorylatable (T194A, S195A, and T194A/S195A [AA]) and phosphorylation mimic (T194D, S195D, and T194D/S195D [DD]) mutants (Table 1). The 110-kDa protein likely represents the mature fully-glycosylated while the 90-kDa protein represents immature core-glycosylated ABCG2 and that ABCG2 biogenesis (expression, trafficking, and maturation) was drastically reduced by mutation of T194 and S195 to Asp. To confirm the above findings, we treated these cells with brefeldin A (BA), which inhibits trafficking of nascent membrane proteins from ER to Golgi [24]. These findings confirm that the positive charges downstream of the C-motif regulate ABCG2 biogenesis and suggest that these positively-charged residues may be physiologically relevant
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