Studies initiated in Robert Herman's lab identified mutations in
sym-3 and
sym-4 as synthetically lethal with loss of
mec-8. Whereas single mutants in
mec-8,
sym-3, and
sym-4 are viable,
mec-8;
sym-4 and
mec-8;
sym-4 double mutants arrest as L1 larvae. We previously reported that
mec-8;
sym-3 and
mec-8;
sym-4 mutants have defects in the embryonic sheath, the apical extracellular matrix surrounding the embryo. We showed that support by the sheath allows the epidermis to resist several intrinsic bio-mechanical forces during morphogenesis including an inward pulling force, generated by the nascent pharynx, as well as circumferential constricting forces. We also showed that FBN-1, a sheath component related to human fibrillins, is critical for epidermal integrity, and that
fbn-1 isoforms are regulated by MEC-8, a conserved splicing factor.
sym-3 encodes a putative membrane-associated protein that is orthologous to human FAM102A.
sym-4 encodes a WD-repeat protein orthologous to human WDR44. The absence of synthetic lethality in
sym-3 sym-4 double mutants reported by Herman and colleagues suggests that SYM-3 and SYM-4 may function together in a complex or pathway. To determine the cellular functions of SYM-3/FAM-102A and SYM-4/WDR44, we carried out IP-LC-MS/MS of GFP-tagged FAM102A and WDR44 in HeLa cells. Notably, these studies identified a physical interaction between WDR44 and FAM102A, as predicted by genetic data. In total, our proteomic approach identified 26 strong FAM102A interactors and 6 strong WDR44 interactors, as well as many interactors that fell below our threshold. Moreover, a number of interactors were identified in both datasets. Strong interactions were detected between WDR44 and two isoforms of RAB11, a conserved Ras-related GTPase that functions in endosomal recycling and exocytosis. These findings are consistent with two previous biochemical studies suggesting that WDR44 may interact with GTP-bound RAB11 (Mammoto et al., 1999; Zeng et al., 1999). Furthermore, SYM-3/FAM102A contains an N-terminal C2 domain, a motif associated with membrane localization found in some Rab11 effectors. Notably, our genetic data are also consistent with RAB-11 functioning in a pathway or complex with the SYMs in C. elegans. Other identified strong interactors include SH3GL1/Endophilin A2, a protein involved in membrane remodeling during endocytosis, and VPS35, a retromer complex component implicated in cargo retrieval and recycling.