Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport.
Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106-289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44-173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met(122) was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met(122) have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.
Beaudoin
Jude
J
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC, Canada J1H 5N4.
Laliberté
Julie
J
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC, Canada J1H 5N4.
Labbé
Simon
S
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC, Canada J1H 5N4.
D020816:Amino Acid Motifs; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008715:Methionine; D000080083:SLC31 Proteins; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*
D027682:Cation Transport Proteins; C509405:Ctr4 protein, S pombe; C430239:Ctr5 protein, S pombe; D005656:Fungal Proteins; D000080083:SLC31 Proteins; D029702:Schizosaccharomyces pombe Proteins; D003300:Copper; D008715:Methionine