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. Author manuscript; available in PMC: 2010 Sep 29.
Published in final edited form as: Biochemistry. 2009 Sep 29;48(38):9122–9131. doi: 10.1021/bi900675v

Figure 2.

Figure 2

Heterodimer of YFP/ABCG2 and E211Q-mutated ABCG2 may be able to transport MTX across the biological membranes. A. YFP/ABCG2 fusion protein mainly forms complex-glycosylated protein at 37 °C. Membrane vesicles containing YFP/ABCG2 fusion protein was digested with endoglycosidase H according to the method described in Experimental Procedures. M, Im and Dg on the right indicate the mature, the core-glycosylated immature and the deglycosylated YFP/ABCG2. B. The relative amounts of ABCG2 proteins in membrane vesicles. Membrane vesicles (1 μg) containing wt ABCG2, YFP/ABCG2, E211Q or YFP/ABCG2 + E211Q were subjected to SDS-PAGE (7%) and probed with ABCG2 mAb BXP-21. The intensities of the ABCG2 protein bands were measured by a scanning densitometer and used to determine the ratios between them. C. Co-expression of YFP/ABCG2 and E211Q in BHK cells forms homodimers and heterodimer. Membrane vesicles containing wt ABCG2 (0.5 μg), YFP/ABCG2 (0.5 μg), E211Q (0.5 μg) or YFP/ABCG2 + E211Q (1 μg) were subjected to SDS-PAGE (4-10% gradient gel) in the absence of reducing agent DTT and probed with ABCG2 mAb BXP-21. The intensities of the ABCG2 protein bands were measured by a scanning densitometer and used to determine the ratios between them. Molecular weight markers are indicated on the left. Mm, Hmd and Htd on the right indicate the monomers of ABCG2, E211Q or YFP/ABCG2, the homodimers of ABCG2, E211Q or YFP/ABCG2 and the heterodimer of YFP/ABCG2 and E211Q. D. E211Q-mutated ABCG2 is unable to hydrolyze ATP. The ATPase assay (in triplicate) was performed according to the method described in Experimental Procedures by using 2 μg of the same membrane vesicles shown in Figure 2B and 2C (1.8862 μg of wtABCG2 + 0.1138 μg of BHK; 1.5145 μg of YFP/ABCG2 + 0.4855 μg of BHK; 2 μg of E211Q; and 0.7918 μg of YFP/ABCG2 + E211Q + 1.2082 μg of BHK) and 4 mM ATP at 37 °C for 30 minutes. After subtraction of the amount of inorganic phosphate generated in the presence of 2 μg BHK membrane vesicles, the velocity of the ATPase activity was calculated and compared to that of ABCG2 (n = 5). E. The heterodimer of YFP/ABCG2 and E211Q may be able to transport MTX across the biological membranes. The MTX uptake assays were carried out, according to the method described in Experimental Procedures, in a 30 μl solution (in triplicate) containing 3 μg of membrane vesicles (2.829 μg of ABCG2 + 0.171 μg of BHK; 2.272 μg of YFP/ABCG2 + 0.728 μg of BHK; 3 μg of E211Q; and 1.188 μg of YFP/ABCG2 + E211Q + 1.812 μg of BHK), 2 mM MTX, 10 mM DTT and 4 mM dATP at 37 °C for 7.5 minutes. After subtraction of the amount of radioactivity bound to the nitrocellulose membrane in the presence of 4 mM AMP from the same sample in the presence of 4 mM dATP, the velocity of the dATP-dependent MTX transport was calculated (n = 4).