The roles of CDF proteins, zinc ions, and SAR-6 in regulating Ras-mediated signaling

A conserved receptor tyrosine kinase-Ras-MAP kinase signaling pathway is required for induction of vulval cell fates in Caenorhabditis elegans . By screening for suppressors of the defects caused by constitutively active let-60 ras, we isolated n2527 and n2508, mutations that define two loci. To clone the affected genes, we developed a method to create local, high-density, single-nucleotide polymorphism maps and positioned n2527 and n2508 to genomic intervals of 9.6 and 11.2 kb, respectively. This high resolution mapping simplified subsequent cloning steps and may facilitate the positional cloning of any gene affected by a mutation in C. elegans. Additionally, this approach may be applicable to other organisms with sequenced genomes.;n2527 affects a previously uncharacterized gene we named cdf-1, because it encodes a member of the cation diffusion facilitator (CDF) family. Loss of cdf-1 reduces Ras signaling during vulval development, suggesting that cdf-1 positively modulates the pathway. The vertebrate CDF protein ZnT-1 functions to reduce cytosolic Zn2+ concentrations. We show that regulation of the Ras pathway is a conserved property of CDF proteins, since expression of ZnT-1 rescues the cdf-1 mutant phenotype and expression of ZnT-1 or CDF-1 promotes Ras signaling in Xenopus oocytes. CDF proteins likely promote Ras signaling by regulating Zn2+, since directly increasing the Zn2+ concentration inhibits Ras signaling. We demonstrate that decreasing cytosolic Zn2+ levels by overexpression of CDF-1 or ZnT-1 enhances Ras signaling, indicating that physiological concentrations of Zn2+ inhibit signaling. We thus propose that Zn2+ maintains the Ras pathway in an inactive state prior to stimulation and that activation of the signaling pathway involves relief of Zn 2+-mediated inhibition.;n2508 affects another previously uncharacterized gene we named sar-6, for s&barbelow;uppressor of a&barbelow;ctivated r&barbelow;as. sar-6 appears to modify Ras signaling during multiple developmental events in C. elegans, acting downstream of or in parallel to ras. sar-6 is likely to encode a novel protein that has a predicted homolog in Drosophila, suggesting that sar-6 function has been conserved throughout evolution. The identification of cdf-1 and sar-6 as modifiers of the Ras pathway demonstrates the utility of forward genetics in unveiling the complex mechanisms that regulate this conserved signaling cascade.