Identification of a Novel High Affinity Copper Transport Complex in the Fission Yeast Schizosaccharomyces pombe

Hao Zhou,Dennis J Thiele

doi:10.1074/jbc.m102004200

Abstract

Copper is an essential nutrient that serves as a co-factor for enzymes involved in critical cellular processes including energy generation, peptide hormone maturation, oxidative stress protection, and iron homeostasis. Although genes have been identified from yeast and mammals encoding a homologous subunit of a plasma membrane high affinity copper transporter, the presence of additional subunits that function as part of a copper transport complex has not been reported. We observed that ctr4(+), a previously identified copper transport protein from the fission yeast Schizosaccharomyces pombe, fails to complement bakers' yeast cells defective in high affinity copper transport and fails to be targeted to the plasma membrane. However, selection for S. pombe genes, which, when co-expressed with Ctr4, confer high affinity copper transport to S. cerevisiae cells resulted in the identification of ctr5(+). Both Ctr4 and Ctr5 are integral membrane proteins, are co-regulated by copper levels and the copper-sensing transcription factor Cuf1, physically associate in vivo, are interdependent for secretion to the plasma membrane, and are each essential for high affinity copper transport. These studies in S. pombe identify Ctr4 and Ctr5 as components of a novel eukaryotic heteromeric plasma membrane complex that is essential for high affinity copper transport.

Highlights

The bakers’ yeast Saccharomyces cerevisiae has been a valuable model system to study eukaryotic copper homeostasis due to its powerful genetics [2, 6]
The S. pombe Ctr4 High Affinity Copper Transporter Is Nonfunctional and Mislocalized in S. cerevisiae—Due to the structural similarity between the S. pombe Ctr4 high affinity copper transporter and the S. cerevisiae Ctr1 and Ctr3 proteins (Fig. 1A, and Ref. 17), we assessed the ability of Ctr4 to complement the inability of an S. cerevisiae ctr1⌬ctr3⌬ strain to grow on nonfermentable carbon sources due to copper insufficiency
The S. pombe ctr4ϩ open reading frame was placed under the control of the strong GPD promoter [29] and S. cerevisiae ctr1⌬ctr3⌬ cells expressing S. pombe Ctr4 were tested for growth on the respiratory carbon sources glycerol/ethanol, for which high affinity copper transport is required for delivery of copper to cytochrome oxidase

Summary

Introduction

The bakers’ yeast Saccharomyces cerevisiae has been a valuable model system to study eukaryotic copper homeostasis due to its powerful genetics [2, 6]. The isolation of genes encoding proteins involved in high affinity copper transport in the fission yeast S. pombe, mice, and humans indicates the presence of variable numbers of amino-terminal Mets motifs and sequence homology to both S. cerevisiae Ctr1 and Ctr3 (2, 16 –18).

Results

Conclusion