Biochemical and Genetic Analyses of Yeast and Human High Affinity Copper Transporters Suggest a Conserved Mechanism for Copper Uptake

Sergi Puig,Jaekwon Lee,Miranda Lau,Dennis J Thiele

doi:10.1074/jbc.m202547200

Sergi Puig, Jaekwon Lee + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m202547200

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Abstract

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.

Highlights

Copper is a transition metal present as a catalytic cofactor in many enzymes including cytochrome c oxidase, Cu,Zn-superoxide dismutase, lysyl oxidase, dopamine ␤-hydroxylase, tyrosinase, or multicopper ferroxidases such as ceruloplasmin in mammals or Fet3 in Saccharomyces cerevisiae
With the exception of Ctr3, the amino-terminal region of copper transporter (Ctr) proteins is rich in motifs containing 3–5 methionine residues arranged as MXXM and MXM, where M represents methionine, and X is any amino acid (Fig. 1, Mets motifs shown in gray)
3 methionine residues are conserved in all known Ctrs: yeast Ctr1 Met-127 residue (Fig. 1, M127 in black characters), which is the last methionine in the Ctr1 Mets motifs and is located 20 amino acids upstream from the putative start of TMD1; and an MXXXM motif located in TMD2 (Fig. 1, MXXXM)

Summary

Introduction

Copper is a transition metal present as a catalytic cofactor in many enzymes including cytochrome c oxidase, Cu,Zn-superoxide dismutase, lysyl oxidase, dopamine ␤-hydroxylase, tyrosinase, or multicopper ferroxidases such as ceruloplasmin in mammals or Fet3 in Saccharomyces cerevisiae. Growth of cells expressing Ctr1 without any Mets motif (Ctr1⌬M1– 8), in ethanol/glycerol, was indistinguishable from cells expressing Ctr1wt (Fig. 3B, YPEG), suggesting that the amino-terminal region of Ctr1 is not absolutely essential for the function of the transporter.

Results

Conclusion