1. Ptc mutations inhibit yeast resistance to doxorubicin and doxorubicin efflux.
Ptc mutations inhibit yeast resistance to doxorubicin and doxorubicin efflux. A, schematic representation of the topologies of Ptc, the Niemann-Pick disease protein (NPC1), and the MDR protein from the Pseudomonas aeroginosa MexD showing a repeated domain composed of 6 transmembrane domains and a hydrophilic loop. B, the GXXXD motif of the putative transmembrane segment 4 is highly conserved in human and drosophila Ptc, in NPC1, and in bacterial transporters of the RND family such as MexB, AcrB, and CzcA. The glycine residue in position 509 and the aspartic acid residue in position 513 were replaced by a valine and a tyrosine, respectively, to produce the mutant protein hPtcG509VD513Y named hPtcVXXXY. C, the mutant protein hPtcVXXXY is fully expressed in yeast. Membranes were prepared from yeasts expressing hPtcWT or hPtcVXXXY, and immunoblotted with antibodies against HA or actin (loading control). D, mutations G509VD513Y inhibit doxorubicin efflux. Yeasts expressing Myo (control), hPtcWT, or hPtcVXXXY were incubated for 2 hours with 10 μmol/L doxorubicin, washed, resuspended in buffer, and centrifuged after 7 minutes. The fluorescence intensity of the supernatants was measured and corrected for loading differences. Shown are mean ± SEM (n = 15, ***P < ). E, mutations G509VD513Y do not significantly inhibit doxorubicin binding. Membranes prepared from yeasts expressing Myo (control), hPtc or hPtcVXXXY were added in 96-well plates containing 500 nmol/L doxorubicin, and time-dependent fluorescence quenching was measured. Shown are mean ± SEM (n = 10). F, mutations G509VD513Y inhibit resistance to doxorubicin. Yeasts expressing Myo (control), hPtcWT, or hPtcVXXXY were grown in normal culture medium or in medium supplemented with 9 or 18 μmol/L doxorubicin. The growth curves reported are representative of 3 independent experiments.
Michel Bidet et al. Mol Cancer Res 2012;10:
©2012 by American Association for Cancer Research