Abstract
BackgroundIn 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 FindingsWe 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 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.SignificanceThese findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability.
Highlights
Given the physiological demands for copper across the biome, both unicellular and multicellular organisms share the requirement for acquiring sufficient levels of copper for cell development and proliferation [1]
The working hypothesis was that the association between the two nonfluorescent Venus carboxyl-terminal fragment (VC)-tagged Ctr4 and Venus amino-terminal fragment (VN)-tagged Ctr5 proteins should trigger the assembly of a functional Venus fluorophore, thereby allowing the detection of a bimolecular fluorescence complementation (BiFC) signal by fluorescence microscopy (Fig. 1C)
Plasmids expressing the VCtagged Ctr4 and VN-tagged Ctr5 proteins were co-transformed into a ctr4D ctr5D strain and the cells were analyzed for respiratory competency as compared to cells transformed with functional Ctr4-GFP and Ctr5-Myc12 proteins [22]
Summary
Given the physiological demands for copper across the biome, both unicellular and multicellular organisms share the requirement for acquiring sufficient levels of copper for cell development and proliferation [1]. An extracellular N-terminal region of variable length contains methionine residues arranged as MX2M and/or MXM motifs (denoted Mets motifs) [5]. Transmembrane span 2 contains a highly conserved MX3M motif that is essential for function in copper transport, whereas transmembrane span 3 possesses a conserved GX3G motif that is required for the trimeric assembly of Ctr molecules [6,7]. An intracellular C-terminal tail of variable length possesses, in general, some cysteine and histidine residues that may be involved in copper binding [1]. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr and Ctr molecules at the cell surface
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