Department of Animal, Plant and Soil Sciences, AgriBio, Centre for AgriBiosciences, 5 Ring Road, La Trobe University, Bundoora, VIC 3086, Australia.
Department of Plant Developmental Biology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany.
J Cell Sci. 2019 Apr 8;132(7):jcs226472. doi: 10.1242/jcs.226472.
The regulation of ion and pH homeostasis of endomembrane organelles is critical for functional protein trafficking, sorting and modification in eukaryotic cells. pH homeostasis is maintained through the activity of vacuolar H-ATPases (V-ATPases) pumping protons (H) into the endomembrane lumen, and counter-action by cation/proton exchangers, such as the NHX family of Na(K)/H exchangers. In plants, V-ATPase activity at the -Golgi network/early endosome (TGN/EE) is important for secretory and endocytic trafficking; however, the role of the endosomal antiporters NHX5 and NHX6 in endomembrane trafficking is unclear. Here we show through genetic, pharmacological and live-cell imaging approaches that double knockout of and results in the impairment of endosome motility and protein recycling at the TGN/EE, but not in the secretion of integral membrane proteins. Furthermore, we report that mutants are partially insensitive to osmotic swelling of TGN/EE induced by the monovalent cation ionophore monensin, and to late endosomal swelling by the phosphatidylinositol 3/4-kinase inhibitor wortmannin, demonstrating that NHX5 and NHX6 function to regulate the luminal cation composition of endosomes.
内质网膜细胞器中离子和 pH 稳态的调节对于真核细胞中功能性蛋白质运输、分拣和修饰至关重要。pH 稳态通过液泡 H+-ATP 酶（V-ATPase）将质子（H）泵入内质网腔来维持，通过阳离子/质子交换器（如 NHX 家族的 Na(K)/H 交换器）进行反向作用。在植物中，高尔基网络/早期内体（TGN/EE）处的 V-ATPase 活性对于分泌和内吞运输很重要；然而，内体逆向转运蛋白 NHX5 和 NHX6 在内膜运输中的作用尚不清楚。通过遗传、药理学和活细胞成像方法，我们发现 和 双敲除会导致内体运动和 TGN/EE 处蛋白质回收受损，但不会影响整合膜蛋白的分泌。此外，我们还报告称， 突变体对半乳糖胺诱导的 TGN/EE 膨胀的单离子载体莫能菌素和磷脂酰肌醇 3/4-激酶抑制剂wortmannin 引起的晚期内体膨胀的敏感性降低，表明 NHX5 和 NHX6 可调节内体腔中的阳离子组成。
