title: Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions.
abstract: The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4-Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X(3)-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4-Ctr5 complex, whereas the Met-X(3)-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC J1H 5N4, Canada.
authors.forename: Jude
authors.initials: J
authors.lastname: Beaudoin
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Research Drive-LSRC-C134, Durham, NC 27710, USA.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Thiele
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC J1H 5N4, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Labbé
authors.affiliation: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), PO Box 73, E-46100 Burjassot, Valencia, Spain.
authors.forename: Sergi
authors.initials: S
authors.lastname: Puig
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D027682:Cation Transport Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D003300:Copper / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D017434:Protein Structure, Tertiary; D000080083:SLC31 Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D027682:Cation Transport Proteins; C509405:Ctr4 protein, S pombe; C430239:Ctr5 protein, S pombe; D000080083:SLC31 Proteins; D029702:Schizosaccharomyces pombe Proteins; D003300:Copper