The C. elegans vulva is a tube consisting of a stack of toroidal cells. Mutation of
egl-26 causes abnormal morphogenesis of the vulF cell at the apex, resulting in a block between the vulval and uterine lumens and an Egl defect. Although vulF adopts a vulE-like morphology in the mutant, there is no vulF to vulE fate transformation. The
egl-26 mutant vulF cell does not express vulE lineage markers and retains the vulF-specific function of inducing
uv1 cells. Additionally, although vulF morphology is abnormal, cell polarity is maintained. Based on lack of expression of a functional EGL-26::GFP fusion in vulF and its presence at the apical membrane of the neighboring vulE cell, we hypothesized that EGL-26 functions non cell-autonomously. To investigate this model, we are preparing an antibody to examine endogenous expression and investigating the molecular mechanism of EGL-26 action. EGL-26 is a member of the NlpC/P60 enzyme family. Isolated
egl-26 mutants have substitutions in the catalytic domains but not specifically at the putative catalytic residues. By creating transgenics carrying EGL-26 with mutations in the catalytic residues, we are testing if catalytic activity is important for vulF morphogenesis. The mammalian family member most related to EGL-26 is LRAT, a palmitoyltransferase that targets the RPE65 protein. The C. elegans genome contains two RPE65-like proteins, F53C3.12 and Y46G5A.24. We demonstrated genetic interactions between F53C3.12 and
egl-26 via RNAi and are labeling wild type and mutant animals with 3H-palmitic acid to determine if the RPE65-like proteins are targets for palmitoylation by EGL-26. LRAT is a membrane protein with two transmembrane domains. Although EGL-26 contains no predicted transmembrane domains, EGL-26::GFP is specifically found at the apical membrane. We are using deletion mutagenesis of EGL-26::GFP to determine the region of EGL-26 that is required for membrane localization. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the
egl-26(
n481) allele causes mis-localization of an EGL-26::GFP fusion. EGL-26(S275F)::GFP is a cytoplasmic protein and cannot rescue an
egl-26 mutant, suggesting that membrane localization is required for function. We are also testing the importance of membrane localization by recruiting EGL-26
(n481)::GFP back to the membrane via addition of alternative membrane localization signals. We expect these experiments to shed light on the molecular and biochemical functions of EGL-26.