Fresquez Theresa, Tam Beatrice M, Eshelman Shannon C, Moritz Orson L, Robichaux Michael A, Deretic Dusanka
Department of Ophthalmology and Visual Sciences, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC V5Z 3N9, Canada.
J Cell Sci. 2025 Jan 15;138(2). doi: 10.1242/jcs.263401. Epub 2025 Jan 23.
The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the expansion of cilia-derived light-sensing organelles, the rod outer segments, via post-Golgi rhodopsin transport carriers (RTCs). Rabin8 (also known as RAB3IP), an effector of Rab11 proteins and a nucleotide exchange factor (GEF) for Rab8 proteins, is phosphorylated at S272 by NDR2 kinase (also known as STK38L), the canine early retinal degeneration (erd) gene product linked to the human ciliopathy Leber congenital amaurosis (LCA). Here, we define the step at which NDR2 phosphorylates Rabin8 and regulates Rab11-to-Rab8 succession in Xenopus laevis transgenic rod photoreceptors expressing human GFP-Rabin8 and its mutants. GFP-Rabin8 accumulated with endogenous Rabin8 at the Golgi-apposed exit sites (GESs), also known as the trans-Golgi network (TGN). Rabin8 mutants deficient in Rab11 binding prevented membrane association of GFP-Rabin8. GFP-Rabin8 and NDR2 kinase both interacted with the RTC-associated R-SNARE VAMP7 at the trans-Golgi and the GESs. Here, GFP-Rabin8 and the phosphomimetic GFP-Rabin8-S272E integrated into RTCs, which were subsequently functionalized by Rabin8 Rab8 GEF activity. Non-phosphorylatable GFP-Rabin8-S272A caused significant GES enlargement and deformation, possibly leading to unconventional membrane advancement toward the cilium, bypassing RTCs. Rabin8 phosphorylation loss due to an NDR2 gene disruption thereby likely causes dysfunctional rhodopsin Golgi-to-cilia trafficking underlying retinal degeneration and early-onset blindness.
Rab11-Rabin8-Rab8纤毛发生复合体通过高尔基体后视紫红质运输载体（RTCs）调节纤毛衍生的光感受器细胞器——视杆外段的扩张。Rabin8（也称为RAB3IP）是Rab11蛋白的效应器和Rab8蛋白的核苷酸交换因子（GEF），在S272位点被NDR2激酶（也称为STK38L）磷酸化，NDR2激酶是犬类早期视网膜变性（erd）基因产物，与人类纤毛病Leber先天性黑蒙（LCA）相关。在这里，我们确定了在非洲爪蟾转基因视杆光感受器中NDR2磷酸化Rabin8并调节Rab11到Rab8转换的步骤，这些光感受器表达人绿色荧光蛋白（GFP）-Rabin8及其突变体。GFP-Rabin8与内源性Rabin8在高尔基体毗邻的出口位点（GESs）（也称为反式高尔基体网络（TGN））聚集。缺乏Rab11结合的Rabin8突变体阻止了GFP-Rabin8与膜的结合。GFP-Rabin8和NDR2激酶在反式高尔基体和GESs处均与RTC相关的R-SNARE VAMP7相互作用。在这里，GFP-Rabin8和模拟磷酸化的GFP-Rabin8-S272E整合到RTCs中，随后通过Rabin8的Rab8 GEF活性发挥功能。不可磷酸化的GFP-Rabin8-S272A导致GES显著增大和变形，可能导致膜绕过RTCs向纤毛进行非常规推进。因此，由于NDR2基因破坏导致的Rabin8磷酸化缺失可能会引起视紫红质从高尔基体到纤毛的运输功能失调，这是视网膜变性和早发性失明的基础。
