Characterization of Oligomeric Human Half-ABC Transporter ATP-binding Cassette G2

Junkang Xu,Yang Liu,Youyun Yang,Susan Bates,Jian-Ting Zhang

doi:10.1074/jbc.m310785200

Junkang Xu, Yang Liu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m310785200

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Abstract

Human ATP-binding cassette G2 (ABCG2, also known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates. Overexpression of human ABCG2 in model cancer cell lines causes multidrug resistance by actively effluxing anticancer drugs. Unlike most of the other ABC transporters which usually have two nucleotide-binding domains and two transmembrane domains, ABCG2 consists of only one nucleotide-binding domain followed by one transmembrane domain. Thus, ABCG2 has been thought to be a half-transporter that may function as a homodimer. In this study, we characterized the oligomeric feature of human ABCG2 using non-denaturing detergent perfluoro-octanoic acid and Triton X-100 in combination with gel filtration, sucrose density gradient sedimentation, and gel electrophoresis. Unexpectedly, we found that human ABCG2 exists mainly as a tetramer, with a possibility of a higher form of oligomerization. Monomeric and dimeric ABCG2 did not appear to be the major form of the protein. Further immunoprecipitation analysis showed that the oligomeric ABCG2 did not contain any other proteins. Taken together, we conclude that human ABCG2 likely exists and functions as a homotetramer.

Highlights

Human ATP-binding cassette G2 (ABCG2, known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates
This observation suggests that the disulfide bond between the ABCG2 subunits was likely formed by oxidation during cell lysis and membrane preparation; it raises a question whether human ABCG2 really forms a homodimer in live cells
Compared with most of other ABC transporters such as multidrug resistance protein 1 (MRP1) and Pgp, ABCG2 is about half the size and is known as a half-transporter

Summary

EXPERIMENTAL PROCEDURES

2– 4 ␮ g of plasma membranes in STBS were mixed with an equal volume of 2ϫ PFO extraction sample buffer (100 mM Tris, pH 8.0, 20% (v/v) glycerol, 0.005% bromphenol blue, 0.5– 8% PFO) followed by addition of dithiothreitol (DTT) to a final concentration of 25 mM and incubated at room temperature for 30 min. Electrophoresis was performed at 140 V at room temperature with running buffer (250 mM glycine and 25 mM Tris, pH 8.3) followed by transfer to polyvinylidene difluoride membrane for Western blot analysis, as described previously [22]. Glycine was added to a final concentration of 2 mM before cells were collected for membrane preparation, followed by separation on a 4 –15% continuous gradient precast gel and Western blot analysis. The final pellet was solubilized in 10 ␮l of sample buffer for SDS-PAGE and autoradiography

RESULTS

Methods of separation

DISCUSSION