Characterization of the FET4 protein of yeast. Evidence for a direct role in the transport of iron

J Biol Chem. 1997 May 2;272(18):11770-7. doi: 10.1074/jbc.272.18.11770.

Abstract

The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requires the FET4 gene. In this report, we present evidence that FET4 encodes the Fe2+ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe2+ substrate. Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent Km and/or Vmax of the low affinity system. These mutations may identify residues involved in Fe2+ binding during transport.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Biological Transport
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cation Transport Proteins
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Copper Transport Proteins
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Regulation, Fungal / drug effects
  • Genes, Fungal
  • Iron / metabolism*
  • Iron / pharmacology
  • Iron-Binding Proteins*
  • Kinetics
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins*
  • Models, Structural
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Protein Structure, Secondary*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*

Substances

  • Carrier Proteins
  • Cation Transport Proteins
  • Copper Transport Proteins
  • FET4 protein, S cerevisiae
  • Iron-Binding Proteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Iron