Copper-dependent trafficking of the Ctr4-Ctr5 copper transporting complex

• Published in: PloS one 2010-08, Vol.5 (8), p.e11964-e11964
• Main Authors: Ioannoni, Raphaël, Beaudoin, Jude, Mercier, Alexandre, Labbé, Simon
• Format: Article
• Language: English
• Subjects: Base Sequence; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cell Biology/Microbial Physiology and Metabolism; Cell Membrane - metabolism; Cell surface; Cell Survival; Complementation; Coordination compounds; Copper; Copper - metabolism; Enzymes; Fluorescence; Genes; Genetic engineering; Genetic recombination; Internalization; Laboratories; Localization; Membranes; Microbiology/Microbial Physiology and Metabolism; Microscopy, Fluorescence; Molecular Biology/Post-Translational Regulation of Gene Expression; Molecular Imaging; Plasma; Protein biosynthesis; Protein interaction; Protein Multimerization; Protein Structure, Quaternary; Protein synthesis; Protein Transport; Protein turnover; Protein-protein interactions; Proteins; Saccharomyces cerevisiae; Schizosaccharomyces - cytology; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Studies; Subfragments; Transcription factors; Venture capital; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0011964

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. 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-Myc(12) displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag(2) 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 internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane. These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability.