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J. Biol. Chem., Vol. 282, Issue 38, 27841-27846, September 21, 2007

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Intramolecular Disulfide Bond Is a Critical Check Point Determining Degradative Fates of ATP-binding Cassette (ABC) Transporter ABCG2 Protein^*

Kanako Wakabayashi¹, Hiroshi Nakagawa, Ai Tamura¹, Shoko Koshiba, Kazuyuki Hoshijima, Masayuki Komada, and Toshihisa Ishikawa²

From the Departments of Biomolecular Engineering and Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A₁, which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.

Received for publication, July 9, 2007 , and in revised form, July 23, 2007.

^* This study was supported by the New Energy and International Technology Development Organization International Joint Research Grant program "International standardization of functional analysis technology for genetic polymorphisms of drug transporters" and research grants (Grants 18201041 and 19659136) from the Japanese Society for the Promotion of Science (JSPS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Japanese Society for the Promotion of Science (JSPS) Research Fellows.

² To whom correspondence should be addressed. Tel: 81-45-924-5800; Fax: 81-45-924-5838; E-mail: tishikaw{at}bio.titech.ac.jp.

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