Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein.
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 A(1), 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.
Wakabayashi
Kanako
K
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Nakagawa
Hiroshi
H
Tamura
Ai
A
Koshiba
Shoko
S
Hoshijima
Kazuyuki
K
Komada
Masayuki
M
Ishikawa
Toshihisa
T
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000737:chemistry; D009363:Neoplasm Proteins / Q000737:chemistry / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D011489:Protein Denaturation; D025801:Ubiquitin / Q000737:chemistry; D015262:Xenobiotics
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D004220:Disulfides; D007976:Leupeptins; D009363:Neoplasm Proteins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D015262:Xenobiotics; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde