The Yeast Arr4p ATPase Binds the Chloride Transporter Gef1p When Copper Is Available in the Cytosol

Abstract

Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys 285 and Cys 288 , is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under ironlimiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, ⌬gef1 cells grow much better than wild type yeast, whereas ⌬arr4 cells are unable to grow. Comparison of the ⌬arr4 with the ⌬arr4⌬gef1 strain suggests that Arr4p antagonizes the function of Gef1p.Copper is an essential metal that is toxic when present in higher concentrations. Homeostatic mechanisms keep copper ions available and protect cellular components against its dangerous redox chemistry. In Saccharomyces cerevisiae, key components of copper homeostasis have been identified (e.g. copper-responsive transcription factors, copper transporters, and copper chaperones) (1). The luminal compartment of the late secretory pathway requires copper for the biosynthesis of copper-containing proteins (2). The endosomal system and the vacuole are thought to contribute to copper storage and detoxification (3-5). Little is known about how cytosolic and luminal copper concentrations of the endomembrane system are integrated in cellular copper ion homeostasis.Gef1p is a CLC chloride channel homologue required for efficient copper supply to the lumen of the Golgi and the prevacuole (6). The recent finding that two of the most closely related mammalian homologues of Gef1p, ClC-4 and -5, exhibit chloride/proton exchange activity (7, 8) like the prokaryotic CLC counterpart (9) suggests that Gef1p may also act as a chloride/proton antiporter. ⌬gef1 mutants show the following phenotypes: loss of high affinity iron uptake, reduced resistance to toxic cations (e.g. high sodium and hygromycin), and sensitivity to alkaline pH of the growth medium (6, 10 -12). High affinity iron uptake is lost because Fet3p (a multicopper oxidase involved in high affinity iron uptake at the cell surface) does not mature normally in ⌬gef1 strains (6).Co...

Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance

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Model for eukaryotic tail-anchored protein binding based on the structure of Get3

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