Yoshimura Shin-Ichiro, Sobajima Tomoaki, Kunii Masataka, Harada Akihiro
Department of Cell Biology, Graduate School of Medicine, The University of Osaka, Suita 565-0871, Osaka, Japan.
Proc Natl Acad Sci U S A. 2025 Jun 3;122(22):e2505975122. doi: 10.1073/pnas.2505975122. Epub 2025 May 30.
Maintenance of pH within membranous organelles is crucial for cellular processes such as posttranslational modifications, ligand-receptor interactions, and proteostasis. The precise mechanisms that determine the luminal pH of each organelle are not fully understood. This study investigated the mechanisms that regulate luminal pH to ensure optimal enzymatic activity. We identified Oxr1 and its paralog Ncoa7, which regulate the vacuolar-type proton pump ATPase (V-ATPase) at the Golgi apparatus and trans-Golgi network (TGN). Oxr1 and Ncoa7 were predominantly localized at the Golgi and TGN membranes, dependent on their binding to various GTP-bound Rab proteins. In vitro experiments using purified recombinant proteins indicated that Oxr1 and Ncoa7 directly bind to the catalytic subunit of V-ATPase, inhibiting its ATP hydrolytic activity via their TLDc domains. We observed significant acidification of the Golgi/TGN lumen in Oxr1- and Ncoa7-depleted cells. Lectin blot analysis demonstrated that depletion of Oxr1 and Ncoa7 led to a defect in protein glycosylation, a major enzymatic posttranslational modification in the Golgi and TGN. Furthermore, depletion of Oxr1 and Ncoa7, along with drug-induced inhibition of glycosylation, increased lysosomal pH and sensitivity to silicon dioxide-induced membrane damage. This apparent lysosomal dysfunction suggested that, in addition to the Golgi and TGN, Oxr1 and Ncoa7 also contribute to the integrity of other organelles. Our findings indicate that Oxr1 and Ncoa7 protect the Golgi and TGN lumen from excess acidification by inhibiting V-ATPase activity and providing an optimal environment for enzymatic activity in the Golgi and TGN.
维持膜性细胞器内的pH值对于细胞过程至关重要，如翻译后修饰、配体-受体相互作用和蛋白质稳态。决定每个细胞器腔内pH值的精确机制尚未完全明确。本研究调查了调节腔内pH值以确保最佳酶活性的机制。我们鉴定出了氧化还原调节因子1（Oxr1）及其旁系同源物核受体辅助激活因子7（Ncoa7），它们在高尔基体和反式高尔基体网络（TGN）中调节液泡型质子泵ATP酶（V-ATPase）。Oxr1和Ncoa7主要定位于高尔基体和TGN膜上，这取决于它们与各种结合GTP的Rab蛋白的结合。使用纯化重组蛋白的体外实验表明，Oxr1和Ncoa7直接与V-ATPase的催化亚基结合，通过其TLDc结构域抑制其ATP水解活性。我们观察到在缺失Oxr1和Ncoa7的细胞中，高尔基体/TGN腔出现明显酸化。凝集素印迹分析表明，缺失Oxr1和Ncoa7会导致蛋白质糖基化缺陷，这是高尔基体和TGN中一种主要的酶促翻译后修饰。此外，缺失Oxr1和Ncoa7，再加上药物诱导的糖基化抑制，会增加溶酶体pH值以及对二氧化硅诱导的膜损伤的敏感性。这种明显的溶酶体功能障碍表明，除了高尔基体和TGN外，Oxr1和Ncoa7也有助于其他细胞器的完整性。我们的研究结果表明，Oxr1和Ncoa7通过抑制V-ATPase活性并为高尔基体和TGN中的酶活性提供最佳环境，保护高尔基体和TGN腔免受过度酸化。
