Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.Iron-sulfur (Fe-S) clusters are cofactors of proteins involved in oxidation-reduction, electron transport, metabolic conversions, and regulatory functions (1). The iron and sulfur are assembled in fixed stoichiometries (e.g. 2Fe-2S, 4Fe-4S) characteristic of the particular protein and coordinated to critical cysteines in the primary peptide backbone (2). Within cells, iron availability for synthesis of iron-sulfur proteins and other biological functions must be tightly regulated, because excess iron is toxic (3). Excess iron leads to free radical reactions that damage membranes, proteins, and DNA (4). Here we describe a regulatory control mechanism that coordinates iron uptake, iron distribution, and the levels of iron-sulfur cluster proteins in the eukaryote Saccharomyces cerevisiae. The regulator responsible for these effects is Nfs1p.Examination of the S. cerevisiae genome data base reveals that Nfs1p is the single yeast homolog of bacterial IscS (5, 6) and NifS (7). There is strong evidence, both biochemical and genetic, showing that the bacterial protein NifS mobilizes sulfur from cysteine and mediates Fe-S cluster assembly. Bacterial mutants of NifS were found to be deficient in the assembly of both Fe protein and MoFe protein subunits of nitrogenase (8, 9). NifS through its enzymatic activity was found to reactivate the apo form of nitrogenase in which the Fe-S cluster was removed by chelation (10). Elegant biochemical work has elucidated this catalytic process: NifS was shown to be a pyridoxal phosphate-containing homodimer that catalyzes the formation of elemental sulfur from L-cysteine (7). A conserved lysine residue in the bacterial NifS protein (equiva...

The yeast frataxin homolog (Yfh1p) participates in mitochondrial iron homeostasis. The phenotypic defects of the Delta yfh1 mutant include drastic accumulation of iron in mitochondria and slow growth. The Yfh1p precursor protein contains two N-terminal domains that are sequentially cleaved by the matrix processing peptidase on import into mitochondria, generating the mature protein. We have precisely mapped these two cleavage sites. Mutations blocking the first or the second cleavage of Yfh1p do not interfere with its in vitro import or with its ability to complement phenotypes of the Delta yfh1 mutant strain. Distinct roles have been ascertained for the two cleaved domains of Yfh1p. The first cleaved domain (domain I) is sufficient for in vitro mitochondrial import of a non-mitochondrial passenger protein. However, neither domain I nor other matrix-targeting signals alone can support efficient in vitro import of mature Yfh1p. The second cleaved domain (domain II) is required as a spacer between a targeting signal and mature Yfh1p. Likewise, when Yfh1p constructs lacking domain I or II are expressed in vivo, they fail to attain appreciable steady-state amounts in mitochondria and cannot complement phenotypes of the Delta yfh1 mutant.