We obtained four additional lines of evidence supporting the notion that CYSL-1 regulates the EGL-9 pathway as a cell-signaling mediator independently of its cysteine synthase activity. First,

the C. elegans genome does not appear to encode any homologs of O-serine acetyltransferase (SAT), which is an obligate component of the cysteine synthase pathway in bacteria and plants ( Mozzarelli et al., 2011 and Wirtz and see more Droux, 2005). BLASTP searches of animal protein databases against bacterial or plant SAT protein queries yielded only three significant hits (E value < 1e-30), in honey bees, Xenopus, and Caenorhabditis remanei, respectively. However, all three lack the invariant C-terminal isoleucine essential for binding to OASS ( Campanini et al., 2005, Francois et al., 2006 and Mozzarelli et al.,

2011), and no other Caenorhabditis species appeared in the search. Second, a potential Fasudil manufacturer bacterial source of OAS as a cysteine synthase substrate for CYSL-1 is unlikely, since feeding rhy-1(n5500) mutants on a cysE-deleted E. coli strain deficient in OAS synthesis did not rescue the rhy-1(n5500) phenotype ( Figure S6H). Third, we found that a lysine in an otherwise highly conserved motif crucial both for binding SATs and for functional CS activity ( Bonner et al., 2005) is a proline in CYSL-1 ( Figure S6G). Fourth, we found that CYSL-1 directly interacts with the C terminus of EGL-9 instead of forming a cysteine synthase complex via its active site, as shown and discussed below. In our rhy-1(n5500) suppressor screen, we isolated over three mutations (n5535, n5539, and n5552) that strongly suppressed K10H10.2::GFP expression and the defective O2-ON response ( Table 1A and Figure 6A). Linkage mapping placed n5535 on the right arm of chromosome V close to egl-9, which prompted us to determine the sequence of the egl-9 coding region of these mutants. We found that n5535 animals carry a missense mutation that converts the EGL-9 C-terminal sequence

EYYI to KYYI, while the n5539 and n5552 alleles alter a splicing donor and a splicing acceptor site, respectively, causing EGL-9 to be prematurely truncated near the EGL-9 C terminus without affecting the O2-sensing proline-hydroxylase domain ( Figure 6B). We noticed that the EYYI sequence of EGL-9 resembles the C-terminal SAT sequence DYVI, which penetrates into the active site of OASS, the CYSL-1 homolog in Arabidopsis ( Francois et al., 2006). These observations, together with the dominant nature of the n5535 phenotype and our epistasis analysis indicating that CYSL-1 inhibits EGL-9, suggested that n5535 might disrupt an EGL-9-interacting interface with CYSL-1 and in that way dominantly suppress rhy-1 LOF phenotypes. To test directly whether CYSL-1 binds to the EGL-9 C terminus, we generated a series of egl-9 mutant constructs and analyzed them in a yeast two-hydrid assay.