copper-dependent trafficking of the ctr4-ctr5 copper transporting complexcopper-dependent贩卖ctr4-ctr5铜运输复杂.pdfVIP

Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex ¨ ´ Raphael Ioannoni, Jude Beaudoin, Alexandre Mercier, Simon Labbe* ´ ´ ´ ´ ´ Departement de Biochimie, Faculte de medecine et des sciences de la sante, Universite de Sherbrooke, Sherbrooke, Canada Abstract Background: In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface. Methodology/Principal Findings: We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4+ and ctr5+ genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc12 displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co- expressed in the presence of Ctr4-Flag2 failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internaliz