Winkley Samuel R, Kane Patricia M
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
J Biol Chem. 2025 Apr;301(4):108381. doi: 10.1016/j.jbc.2025.108381. Epub 2025 Mar 4.
V-ATPases are highly conserved ATP-driven rotary proton pumps found widely among eukaryotes that are composed of two subcomplexes: V and V. V-ATPase activity is regulated in part through reversible disassembly, during which V physically separates from V and both subcomplexes become inactive. Reassociation of V to V reactivates the complex for ATP-driven proton pumping and organelle acidification. V-ATPase reassembly in Saccharomyces cerevisiae requires the RAVE complex (Rav1, Rav2, and Skp1), and higher eukaryotes, including humans, utilize the Rabconnectin-3 complex. Mammalian Rabconnectin-3 has two subunits: Rabconnectin-3α and Rabconnectin-3β. Rabconnectin-3α isoforms are homologous to Rav1, but there is no known Rav2 homolog, and the molecular basis of the interaction between the Rabconnectin-3α and β subunits is unknown. We identified ROGDI as a Rav2 homolog and novel Rabconnectin-3 subunit. ROGDI mutations cause Kohlschutter-Tonz syndrome, an epileptic encephalopathy with amelogenesis imperfecta that has parallels to V-ATPase-related disease. ROGDI shares extensive structural homology with yeast Rav2 and can functionally replace Rav2 in yeast. ROGDI binds to the N-terminal domains of both Rabconnectin-3 α and β, similar to Rav2 binding to Rav1. Molecular modeling suggests that ROGDI may bridge the two Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 subunits from detergent-solubilized lysates and is present with them in immunopurified lysosomes of mammalian cells. In immunofluorescence microscopy, ROGDI partially localizes with Rabconnectin-3α in acidic perinuclear lysosomes. The discovery of ROGDI as a novel Rabconnectin-3 interactor sheds new light on both Kohlschutter-Tonz syndrome and the mechanisms behind mammalian V-ATPase regulation.
V-ATP酶是高度保守的由ATP驱动的旋转质子泵，广泛存在于真核生物中，由两个亚复合体组成：V和V。V-ATP酶的活性部分通过可逆解离来调节，在此过程中V与V物理分离，两个亚复合体均失去活性。V与V重新结合会使复合体重新激活，以进行由ATP驱动的质子泵浦和细胞器酸化。酿酒酵母中的V-ATP酶重新组装需要RAVE复合体（Rav1、Rav2和Skp1），包括人类在内的高等真核生物则利用Rabconnectin-3复合体。哺乳动物的Rabconnectin-3有两个亚基：Rabconnectin-3α和Rabconnectin-3β。Rabconnectin-3α亚型与Rav1同源，但尚无已知的Rav2同源物，且Rabconnectin-3α和β亚基之间相互作用的分子基础尚不清楚。我们鉴定出ROGDI是一种Rav2同源物和新型Rabconnectin-3亚基。ROGDI突变会导致科尔施胡特-通兹综合征，这是一种伴有牙釉质发育不全的癫痫性脑病，与V-ATP酶相关疾病有相似之处。ROGDI与酵母Rav2具有广泛的结构同源性，并且在酵母中可以在功能上替代Rav2。ROGDI与Rabconnectin-3α和β的N端结构域结合，类似于Rav2与Rav1的结合。分子模型表明，ROGDI可能连接两个Rabconnectin-3亚基。ROGDI与去污剂溶解的裂解物中的Rabconnectin-3亚基进行共免疫沉淀，并与它们一起存在于哺乳动物细胞的免疫纯化溶酶体中。在免疫荧光显微镜检查中，ROGDI部分定位于酸性核周溶酶体中的Rabconnectin-3α。ROGDI作为新型Rabconnectin-3相互作用蛋白的发现，为科尔施胡特-通兹综合征以及哺乳动物V-ATP酶调节背后的机制提供了新的线索。
