title: An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry.
abstract: As a cellular organelle, the cilium contains a unique protein composition. Entry of both membrane and cytosolic components is tightly regulated by gating mechanisms at the cilium base; however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs), which comprise a ciliary pore complex (CPC). To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC), for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated (1) active transport of BSA into the nuclear compartment and (2) the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore-clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore, whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle's limiting membrane.
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takao
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Dishinger
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: H Lynn
authors.initials: HL
authors.lastname: Kee
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Justine M
authors.initials: JM
authors.lastname: Pinskey
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Ben L
authors.initials: BL
authors.lastname: Allen
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address: kjverhey@umich.edu.
authors.forename: Kristen J
authors.initials: KJ
authors.lastname: Verhey
mesh_terms: D000818:Animals; D002923:Cilia / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D051379:Mice; D041681:NIH 3T3 Cells; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D028861:Nuclear Pore Complex Proteins