ABCMdb

PMID: 17686774

Wakabayashi K, Nakagawa H, Tamura A, Koshiba S, Hoshijima K, Komada M, Ishikawa T
Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein.
J Biol Chem. 2007 Sep 21;282(38):27841-6. Epub 2007 Aug 8., 2007-09-21 [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly 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. Login to comment 26 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly When both Cys-592 and Cys-608 were substituted to Gly, the resulting C592G/C608G variant could form a homodimer, but its expression level was remarkably reduced. Login to comment 35 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly 38, pp. 27841-27846, September 21, 2007 (c) 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cys Role in ABCG2 Protein Stability Furthermore, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was not detectable (10). Login to comment 40 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly EXPERIMENTAL PROCEDURES Cell Culture-Flp-In-293 cells expressing ABCG2 WT or Cys-to-Gly variants (C592G and C608G) were established and cultured as described previously (10). Login to comment 58 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly RESULTS AND DISCUSSION Protein Expression Levels of ABCG2 WT, C592G, and C608G in Flp-In-293 Cells-We investigated the role of disulfide bond formation in the protein stability and cellular localization of ABCG2. Login to comment 64 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly B, expression of ABCG2 WT, C592G, and C608G in Flp-In-293 cells at the transcription and protein level. Login to comment 65 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly The transcription level was analyzed by reverse transcription-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment 68 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly DataareexpressedasmeanvaluesϮS.D.intriplicateexperiments.C,enhancementoftheproteinlevelofABCG2variantsbyMG132treatment.Flp-In-293cells expressing ABCG2 WT, C592G, or C608G were incubated with 2 ␮M MG132 for 0, 24, and 48 h. ABCG2 protein levels were analyzed by immunoblotting after PNGase F treatment. Login to comment 79 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly A, immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment 81 ABCG2 p.Cys592Gly Horizontal bars correspond to 20 ␮m. B, effect of MG132 on ubiquitination of ABCG2 proteins.Afterincubationwithorwithout2␮M MG132for24h,celllysatesampleswerepreparedfromFlp-In-293cellsexpressingABCG2WT,C592G,orC608G. Login to comment 83 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly In the present study, we prepared two ABCG2 variants, C592G and C608G, in which Cys-592 and Cys-608 in the extracellular loop were substituted to Gly, respectively (Fig. 1A). Login to comment 85 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Accordingly, as demonstrated in Fig. 1B, ABCG2 mRNA levels detected by reverse transcription-PCR were almost equal among ABCG2 WT, C592G, and C608G. Login to comment 86 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly However, substitution of either Cys-592 or Cys-608 to glycine resulted in a significant decrease in protein levels of ABCG2 in Flp-In-293 cells (Fig. 1B). Login to comment 87 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Both C592G and C608G variants formed homodimers (data not shown; see Fig. 8 in Ref. 10). Login to comment 88 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Immunoblotting without PNGase F treatment revealed two faint bands for each of the C592G and C608G variants, and these bands corresponded to glycosylated (MW ϭ 81,000) and non-glycosylated (MW ϭ 63,000) forms (Fig. 1B). Login to comment 90 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly After PNGase F treatment, one single band (MW ϭ 63,000) was detected not only for ABCG2 WT but also for the C592G and C608G variants (Fig. 1B). Login to comment 91 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly It is suggested that N-linked glycosylation was formed in the C592G and C608G variants as well as the WT in Flp-In-293 cells. Login to comment 92 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Effect of MG132 or Bafilomycin A1 on the Protein Levels of ABCG2 WT, C592G, and C608G-As shown in Fig. 1C, the protein levels of those ABCG2 variants were remarkably enhanced by the treatment with the proteasome inhibitor MG132. Login to comment 94 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly The increases in protein levels of the ABCG2 C592G and C608G variants occurred in a time-dependent manner during the MG132 treatment. Login to comment 97 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly On the other hand, as depicted in Fig. 1D, the protein level of ABCG2 WT increased more than 5-fold when cells were treated with bafilomycin A1, which inhibits lysosomal degradation, whereas the C592G or C608G variants were little affected by the same treatment. Login to comment 99 ABCG2 p.Cys608Gly ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly ABCG2 p.Cys592Gly Effect of MG132 on the Cellular Localization and Function of ABCG2 WT, C592G, and C608G-It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization and functional activity of the ABCG2 C592G and C608G variants. Login to comment 100 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Fig. 2A depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G that were incubated with or without 2 ␮M MG132 for 24 h. ABCG2 proteins were probed with theBXP-21antibodyandthenlabeledwithgreenfluorescencedye (Alexa Fluor 488), whereas DNA in the nuclei was stained with propidium iodide (red fluorescence). Login to comment 102 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly A remarkable difference was observed after the MG132 treatment, however, in terms of the cellular localization of the C592G or C608G variant proteins. Login to comment 103 ABCG2 p.Cys592Gly Without MG132 treatment, immunofluorescence of the C592G orC608Gvariantswasextremelyweakattheplasmamembraneof Flp-In-293 cells (Fig. 2A). Login to comment 104 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Rather, those variant proteins were detected in intracellular compartments, suggesting that the sorting of C592G or C608G variants to the plasma membrane domain was impaired. Login to comment 105 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly It is noteworthy, however, that the plasma membrane localizations of the C592G and C608G variants were clearly detected after the MG132 treatment (Fig. 2A). Login to comment 106 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly To investigate the effect of MG132 on the ubiquitinated state of ABCG2 proteins, the Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G were incubated in the presence or absence of 2 ␮M MG132 for 24 h. Login to comment 107 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly As demonstrated in Fig. 2B, significant increases in the ubiquitinated forms of the C592G and C608G variant proteins were detected by immunoblotting with the anti-ubiquitin antibody after immunoprecipitation with the anti-ABCG2 antibody BXP-21 (upper panel). Login to comment 111 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Based on these results, it is suggested that both C592G and C608G variants that were sorted to the plasma membrane domain after the MG132 treatment were functionally active to extrude SN-38 out of the cells. Login to comment 117 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly C, drug resistance profiles of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment 122 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly D, schematic illustration for plausible pathways for protein processing and degradation of ABCG2 WT and Cys-to-Gly mutated variants (C592G and C608G). Login to comment 136 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly Misfolded proteins (e.g. ABCG2 C592G and C608G variants) may be removed from the ER by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system (24). Login to comment 142 ABCG2 p.Cys608Gly ABCG2 p.Cys592Gly In addition, we anticipate that some part of ubiquitinated ABCG2 C592G and C608G variants can undergo deubiquitination and subsequently be sorted to the plasma membrane domain (Fig. 2D) since these variants were markedly localized at the plasma membrane domain after MG132 treatments (Fig. 2A). Login to comment