Abstract
ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.
Highlights
Resistance of cancers to a chemically broad spectrum of drugs is referred to as multidrug resistance (MDR)
YFP fragments supports protein localization and function The dimerization of ABCG2 was investigated using bimolecular fluorescence complementation, a protein:protein interaction technique that relies on the ability of molecular fragments of YFP to re-associate and refold into a fluorescent structure (Figure 1A [31])
ABCG2 has both its N-terminus and its Cterminus exposed to the intracellular surface [32] and 6 constructs were required for use in this study (Figure 1B)
Summary
Resistance of cancers to a chemically broad spectrum of drugs is referred to as multidrug resistance (MDR). We show that ABCG2 was trafficked to the cell membrane and functional when tagged N-terminally with half-molecules of a variant form of YFP with efficient refolding kinetics
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