Targeting of Nphp3 to the primary cilia is controlled by an N‐terminal myristoylation site and coiled‐coil domains

Abstract

Primary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1-11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled-coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences (CTSs) are unknown. In the present study, we generated a series of GFP-tagged deletion constructs of Nphp3 and tried to find the CTSs of Nphp3. We found that the N-terminal 201 amino acid fragment (Nphp3 [1-201]), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N-terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N-terminal fragments, Nphp3 (8-201), Nphp3 (52-201), and Nphp3 (96-201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two-step process.

Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition

“…Unc119b recognizes the myristoylated amino-terminal residues of both Nphp3 and Cystin, thereby protecting them from interaction with the lipid bilayer (Wright et al 2011). Only when bound to cargo can Unc119b enter the cilium across the TZ, where it encounters Arl3, a small GTP-binding protein that binds to and induces the dissociation of the Unc119-cargo complex, thereby allowing the myristoylated cargo to associate with the ciliary membrane (Wright et al 2011; Zhang et al 2011; Nakata et al 2012). In turn, Arl3 activity is regulated by an Arl3-GAP (RP2) and an Arl3-GEF (Arl13b), which regulate its association with GDP or GTP and, therefore, its activity (Wright et al 2011; Gotthardt et al 2015).…”

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not cleavage at GPS site

“…The PC1-CTT contains more than one CTS An N-terminal fragment containing two coiled-coil motifs has been shown to be necessary and sufficient to target Nphp3 to the primary cilia, functioning as a CTS (Nakata et al, 2012). We next tested whether the coiled-coil motif in the PC1-CTT or the PC1-CTT itself is sufficient to target foreign proteins to the cilia.…”

From the cytoplasm into the cilium: Bon voyage

“…The N-terminal myristoylation motif of NPHP3 is necessary for its ciliary localization 188 . This sequence is, however, insufficient for ciliary targeting, which indicates that additional signals in the NPHP3 polypeptide contribute to ciliary localization 188 .…”

“…The N-terminal myristoylation motif of NPHP3 is necessary for its ciliary localization 188 . This sequence is, however, insufficient for ciliary targeting, which indicates that additional signals in the NPHP3 polypeptide contribute to ciliary localization 188 . An additional layer of complexity in the ciliary targeting of NPHP3 has been revealed by observations that an N-terminal coiled-coil domain of NPHP3, which does not include the myristoylation site, is both necessary and sufficient for targeting to the ciliary centriole.…”

“…In photoreceptor cells, it may also function as a solubilization factor during cytoplasmic transport of the ciliary Transducin 186 . Finally, NPHP3 transport seems to proceed independently from the ciliary pore Ran/Importin system 188 . Thus, lipidated proteins appear to follow their own unique traffic rules during cilia-directed transport.…”