Rab8 and TNPO1 are ciliary transport adaptors for GTPase Arl13b by interacting with its RVEP motif containing ciliary targeting sequence.

Arl13b, an ARF/Arl-family GTPase, is highly enriched in the cilium. Recent studies have established Arl13b as one of the most crucial regulators for ciliary organization, trafficking, and signaling. The ciliary localization of Arl13b is known to require the RVEP motif. However, its cognate ciliary transport adaptor has been elusive. Here, by imaging the ciliary localization of truncation and point mutations, we defined the ciliary targeting sequence (CTS) of Arl13b as a C-terminal stretch of 17 amino acids containing the RVEP motif. We found Rab8-GDP, but not Rab8-GTP, and TNPO1 simultaneously and directly bind to the CTS of Arl13b in pull-down assays using cell lysates or purified recombinant proteins. Furthermore, Rab8-GDP substantially enhances the interaction between TNPO1 and CTS. Additionally, we determined that the RVEP motif is an essential element as its mutation abolishes the interaction of the CTS with Rab8-GDP and TNPO1 in pull-down and TurboID-based proximity ligation assays. Finally, the knockdown of endogenous Rab8 or TNPO1 decreases the ciliary localization of endogenous Arl13b. Therefore, our results suggest Rab8 and TNPO1 might function together as a ciliary transport adaptor for Arl13b by interacting with its RVEP-containing CTS.

Mechanisms of ciliary targeting: entering importins and Rabs.

Primary cilium is a rod-like plasma membrane protrusion that plays important roles in sensing the cellular environment and initiating corresponding signaling pathways. The sensory functions of the cilium critically depend on the unique enrichment of ciliary residents, which is maintained by the ciliary diffusion barrier. It is still unclear how ciliary cargoes specifically enter the diffusion barrier and accumulate within the cilium. In this review, the organization and trafficking mechanism of the cilium are compared to those of the nucleus, which are much better understood at the moment. Though the cilium differs significantly from the nucleus in terms of molecular and cellular functions, analogous themes and principles in the membrane organization and cargo trafficking are notable between them. Therefore, knowledge in the nuclear trafficking can likely shed light on our understanding of the ciliary trafficking. Here, with a focus on membrane cargoes in mammalian cells, we briefly review various ciliary trafficking pathways from the Golgi to the periciliary membrane. Models for the subsequent import translocation across the diffusion barrier and the enrichment of cargoes within the ciliary membrane are discussed in detail. Based on recent discoveries, we propose a Rab-importin-based model in an attempt to accommodate various observations on ciliary targeting.

The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex.

We report here an image-based method to quantify the stoichiometry of diffraction-limited sub-cellular protein complexes in vivo under spinning disk confocal microscopy. A GFP single molecule fluorescence standard was first established by immobilizing His-tagged GFP molecules onto the glass surface via nickel nitrilotriacetic acid functionalized polyethylene glycol. When endogenous nucleoporins were knocked down and replaced by the exogenously expressed and knockdown-resistant GFP-nucleoporins, the stoichiometry of the nucleoporin was estimated by the ratio of its fluorescence intensity to that of the GFP single molecules. Our measured stoichiometry of Nup35, Nup93, Nup133 and Nup88 is 23, 18, 14 and 9 and there are possibly16 copies of Nup107-160 complex per nuclear pore complex.

A ternary complex comprising transportin1, Rab8 and the ciliary targeting signal directs proteins to ciliary membranes.

The sensory functions of cilia are dependent on the enrichment of cilium-resident proteins. Although it is known that ciliary targeting signals (CTSs) specifically target ciliary proteins to cilia, it is still unclear how CTSs facilitate the entry and retention of cilium-resident proteins at the molecular level. We found that non-ciliary membrane reporters can passively diffuse into cilia through the lateral transport pathway, and the translocation of membrane reporters through the ciliary diffusion barrier is facilitated by importin binding motifs and domains. Screening known CTSs of ciliary membrane residents uncovered that fibrocystin, photoreceptor retinol dehydrogenase, rhodopsin and retinitis pigmentosa 2 interact with transportin1 (TNPO1) through previously identified CTSs. We further discovered that a new ternary complex, comprising TNPO1, Rab8 and a CTS, can assemble or disassemble under the guanine nucleotide exchange activity of Rab8. Our study suggests a new mechanism in which the TNPO1-Rab8-CTS complex mediates selective entry into and retention of cargos within cilia.