Cystin Localizes to Primary Cilia via Membrane Microdomains and a Targeting Motif

Abstract

Primary cilia are dynamic, complex structures that contain Ͼ500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1 cpk mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145-amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein-tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.

An ARL3–UNC119–RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium

“…In contrast to our N-terminally tagged construct (data not shown), the C-terminally tagged NPHP3 was able to localize to the primary cilium (Fig. 1A), suggesting, as seen for other ciliary proteins, including cystin (Tao et al 2009), that myristoylation aids in proper NPHP3 targeting. Several attempts to generate a nonmyristoylated, G2A mutant NPHP3 stable cell line suitable for tandem affinity purification were unsuccessful, likely due to poor stability of the mutant protein.…”

“…These included the known ciliary protein cystin, which is mutated in the mouse cystic kidney model cpk (Hou et al 2002). Cystin also requires myristoylation for proper ciliary membrane localization (Tao et al 2009). To validate the interaction with cystin in cells, we generated a stable RPE cell line expressing the first 20 amino acids of cystin tagged on its C terminus, which is sufficient for targeting cystin to the cilium (Supplemental Fig.…”

Cystin, Cilia, and Cysts

“…Tao et al 8 test this hypothesis by in vivo labeling and indeed prove this is the case. Furthermore, they demonstrate this modification is necessary for association of cystin with membrane microdomains/lipid rafts.…”

“…Of particular interest is the observation that detergent extraction of trypanosome flagella reveals detergent-resistant membrane patches approximating the size of intraflagella transport particles. 13 This finding suggests transmembrane proteins traffic in the cilium through their clustering into membrane microdomains coupled to core intraflagella transport machinery; however, Tao et al 8 demonstrate that myristoylation is necessary but not sufficient to target cystin to the cilium, indicating a second trafficking determinant is required.…”

“…[5][6][7] In photoreceptors, this motif binds the small G-protein, Arf4, and in conjunction with Rab11, FIP3, and ASAP1 promotes budding of ciliary-destined cargo from the transgolgi network. 7 In this issue of JASN, Tao et al 8 identify a novel ciliary trafficking determinant in the cystoprotein cystin that furthers our understanding of how proteins are selectively targeted to the cilium. This group previously identified cystin as the gene disrupted in the cpk (congenital polycystic kidney) mouse model of autosomal recessive PKD.…”