Informatic, biochemical, and functional analysis implicating lectins as FAMK-1 substrates. (A) Schematic highlighting that FAMK-1, which targets serines two residues before a glutamate or a phosphoserine, is predicted to phosphorylate serine clusters preceding two glutamates. The boxed workflow indicates the proteome search approach that identified lectins as potential FAMK-1 substrates. Signal peptide and membrane topology prediction was performed using SPOCTOPUS and SMART. (B) Schematic of experimental protocol employed to test if identified lectins are FAMK-1 substrates. Autoradiograms and blots below show analysis for three candidate lectin substrates; the sequence stretches likely to be targets of phosphorylation are shown above the gels. (C) Analysis of CLEC-233, conducted as in B. KDEL-containing FAMK-1 was also analyzed for its ability to phosphorylate CLEC-233 (panels on the right). (D) Embryonic lethality and fertility analysis for the indicated conditions; note that this analysis was conducted in the presence of transgenes expressing fluorescent fusions that mark the plasma membrane and chromatin (transgenes from strain OD95; Green et al., 2011). Error bars are the SD; *****, P < 0.0001 (t test). (E) Images of late-stage embryos genome edited to mutate the two glutamates (clec-233(EE>AA)). Arrows point to multinucleation events. Scale bars, 5 µm.