Takao Daisuke, Dishinger John F, Kee H Lynn, Pinskey Justine M, Allen Ben L, Verhey Kristen J
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Curr Biol. 2014 Oct 6;24(19):2288-94. doi: 10.1016/j.cub.2014.08.012. Epub 2014 Sep 25.
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.
作为一种细胞器，纤毛含有独特的蛋白质组成。膜成分和胞质成分进入纤毛均受到纤毛基部门控机制的严格调控；然而，纤毛门控的具体机制在很大程度上仍不清楚。我们之前提出，胞质成分的进入是由类似于核转运的机制调控的，并且依赖于核孔蛋白（NUPs），这些核孔蛋白组成了纤毛孔复合体（CPC）。为了研究纤毛门控机制，我们开发了一种通过强制二聚化抑制NUP功能来堵塞孔道的系统。我们将核孔复合体（NPC）中央通道的一个成分NUP62作为靶点，通过用同型二聚化的Fv结构域标记它来进行强制二聚化。作为原理验证，我们表明NUP62-Fv的强制二聚化减弱了（1）牛血清白蛋白（BSA）向核区室的主动转运以及（2）驱动蛋白-2马达蛋白KIF17向纤毛区室的转运。使用孔道堵塞技术，我们发现NUP62的强制二聚化减弱了胞质蛋白的门控进入，但不影响膜蛋白的进入或小胞质蛋白的扩散进入。我们提出了一个模型，其中胞质蛋白向核区室和纤毛区室的主动转运需要中央孔的功能性NUPs，而膜蛋白的侧向进入利用了一种可能特定于每个细胞器限制膜的不同机制。
