The morphogenesis of the C. elegans male gonad occurs by the migration of the individual linker cell and the adhesion of the proliferating follower cells behind this leader. We identified a conserved, single-pass transmembrane protein, TAG-256, that is required for the gonadal cells to stay connected during their migration; in mutants with a partial
tag-256 deletion, dissociated gonadal cells are left behind during the migration, and this mutant can be rescued with a wild-type genomic copy of
tag-256. TAG-256::YFP fusion protein expresses in the entire somatic gonad and localizes to the plasma membrane. Antibodies generated to different domains of TAG-256 show additional localization of the extracellular domain to large cytoplasmic vesicles and the intracellular domain to the nucleus, suggesting proteolytic cleavage. Since TAG-256 has no known interactors, we used SILAC mass spectrometry on human 293T cells to identify protein interactors of its human ortholog, ITFG1. Sixty-six proteins were ³ 5-fold enriched in immunoprecipitates with ITFG1, and of these, 46 proteins (70%) were conserved in C. elegans. To identify the genes that function together with
tag-256 in C. elegans, we performed RNAi of the 46 interactors having worm orthologs in wild-type animals, and looked for a gonad detachment phenotype.
ruvb-1,
ruvb-2 and
tba-2 yielded the same phenotype as
tag-256, and we confirmed the physical interaction of their human orthologs by Western blot. We are currently investigating the subcellular localization of this new complex and the role of the AAA+ ATPases, RUVB-1 and RUVB-2, in this process. Based on the membrane localization of TAG-256 and gonad detachment phenotype of the mutant, we propose that TAG-256 has a physical role in cell-cell adhesion.