Evolutionary proteomics uncovers ciliary signaling components

Monika Abedin Sigg; Tabea Menchen; Jeffery Johnson; Chanjae Lee; Semil P. Choksi; Galo Garcia; Henriette Busengdal; Gerard Dougherty; Petra Pennekamp; Claudius Werner; Fabian Rentzsch; Nevan Krogan; John B. Wallingford; Heymut Omran; Jeremy F. Reiter

To begin to assess the subcellular localization of sea urchin, sea anemone and 388 choanoflagellate ENKUR, we GFP-tagged ENKUR and analyzed its localization in ciliated 389 mammalian cells. We found that GFP-tagged sea urchin and choanoflagellate ENKUR proteins 390 localized to cilia if expressed in mammalian cells ( Figure 5E and F). To determine if ENKUR 391 localizes to cilia in vertebrates, we expressed GFP-ENKUR in X. laevis embryos and found that 392 it localized to the epidermal cell motile cilia ( Figure 5G ). Similarly, mouse ENKUR localized to 393 the motile cilia of tracheal epithelial cells ( Figure 5H and S5C) and the mammalian ortholog of 394 an ENKUR-related protein, ENKD1, localized predominantly to the base of cilia ( Figure 395 S5D). To further analyze ENKUR expression in the mouse, we utilized Enkur -/mice. Western 396 blot corroborated the ENKUR immunofluorescent staining, indicating that the protein is 397 expressed in trachea, and is absent from the trachea of Enkur -/mice ( Figure 5I ). 398