Identification of particular defects in the trafficking of polymorphic ABCG2 variants using a novel, dynamic method

Abstract

ABCG2 (ATP-Binding Cassette, subfamily G, member 2) is a plasma membrane glycoprotein, which actively transports endo- and xenobiotics from the cells. Urate is also a physiologically relevant substrate of ABCG2; thus, dysfunction of the transporter, including trafficking defects, may lead to hyperuricemia or gout. Intracellular localization of proteins is most frequently studied by antibody staining of fixed samples, which can only reflect their steady state distribution. In our study, the cellular routing of the wild type and two naturally occurring polymorphic variants (Q141K and M71V) of ABCG2 was followed by a novel, dynamic approach, the Retention Using Selective Hooks (RUSH) method. On the basis of the RUSH system, we developed a quantitative method to assess the trafficking kinetics of ABCG2 variants in particular subcellular compartments. We found that both Q141K- and M71V-ABCG2 have a major trafficking defect with similar characteristics: a substantial portion of these variants is retained in the ER, and their delivery to the plasma membrane is also deferred. In addition, we studied the effect of various pharmacological agents that potentially promote the plasma membrane delivery of ABCG2 variants. Our results revealed not only the particular defects of the polymorphic ABCG2 variants but also provided an experimental tool for screening drugs that abrogate trafficking impairment, assisting the development of more effective and personalized therapies for gout patients.