A conserved receptor tyrosine kinase-Ras-MAP kinase signal transduction cascade is required for specification of the C. elegans vulva. In a screen for suppressors of the multivulva (Muv) phenotype caused by a constitutively active
let-60 ras gene, we identified the strong loss-of-function mutation
n2527 . The
n2527 mutation causes a weak vulvaless phenotype; epistasis experiments suggest that it functions downstream of
let-60 and upstream of
lin-1 if the genes act in a linear pathway. To positionally clone the gene affected by
n2527 , we created a local, high-density, single-nucleotide polymorphism map and positioned
n2527 within a 9.6-kb interval. The molecular lesion was identified by sequencing, and the candidate gene was confirmed by transformation rescue. The
n2527 mutation creates a premature STOP codon in a previously uncharacterized gene that we named
cdf-1, since it encodes a predicted protein with significant similarity to members of the cation diffusion facilitator (CDF) family. CDF-1 is similar to mammalian ZnT-1 which reduces the intracellular concentration of Zn 2+ ions. CDF proteins have not been previously shown to be involved in signal transduction. We investigated if regulation of Ras signaling is an evolutionarily conserved property of CDF proteins. When expressed under the control of the
cdf-1 promoter, rat ZnT-1 rescues the
cdf-1(
n2527) suppression of
let-60(gf) ras Muv phenotype. Thus a vertebrate CDF protein can function in worms to promote Ras signaling. We also tested if CDF-1 could function in vertebrate signal transduction using Xenopus laevis oocytes. In a sensitized background, overexpression of CDF-1 induces MAP kinase phosphorylation and Xenopus oocyte meiotic maturation, suggesting that worm CDF-1 can stimulate Ras signaling in a vertebrate. To determine if
cdf-1 affects the transport of a heavy metal ion, we grew worms in the presence of additional metal ion.
cdf-1 mutants were hypersensitive to Zn 2+ , as indicated by a reduced growth rate, increased larval lethality and increased sterility compared with wild-type worms. This phenotype is specific to zinc ions, since the growth of
cdf-1 mutants on Co 2+ , Cd 2+ , or Cu 2+ was similar to wild-type. Thus,
cdf-1 is likely to play a widespread role in Zn 2+ metabolism and the analysis of this locus is likely to illuminate how C. elegans regulates Zn 2+ . Because CDF-1 is likely to reduce the intracellular concentration of zinc ions and positively modulate Ras signaling, we hypothesized that zinc ions inhibit Ras signaling. To test this hypothesis, we analyzed the phenotype of Ras pathway mutants exposed to increased levels of Zn 2+ in the media. Extra zinc reduces the penetrance of the Muv phenotype caused by gain-of-function mutations in the
let-60 ras and
let-23 genes while the
lin-1(lf) Muv phenotype was unaffected. The effect of increased zinc ions on these mutants phenocopies that of
cdf-1 mutants and suggests that there is a zinc-sensitive pathway component downstream of
let-60 ras and upstream of
lin-1. We propose a model in which zinc ions inhibit signal transduction and that CDF proteins promote Ras signaling by reducing the intracellular concentration of this negative regulator. This regulatory circuit is likely to be conserved.