Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Function (Oxf). 2024 Jul 11;5(4). doi: 10.1093/function/zqae025.
The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.
质子泵 V-ATPase 驱动着重要的生物过程，如细胞内细胞器的酸化。至关重要的是，V-ATPase 的 V1 和 VO 结构域必须组装在一起才能产生有功能的全酶。V-ATPase 功能障碍会导致癌症、神经退行性疾病和糖尿病，以及由于质子分泌肾闰细胞（IC）活性降低导致的全身性酸中毒。然而，人们对哺乳动物 V-ATPase 的分子调节知之甚少。我们鉴定了一种新型的哺乳动物 V-ATPase 相互作用蛋白，果蝇 X 染色体基因样 1（Dmxl1），又名 Rabconnectin-3A。Dmxl1 的酵母同源物 Rav1p 是一种复合物的一部分，该复合物可催化结构域的可逆组装。因此，我们假设 Dmxl1 是一种哺乳动物 V-ATPase 组装因子。在这里，我们生成了肾脏 IC 特异性 Dmxl1 敲除（KO）小鼠，它们的尿液 pH 值很高，与 B1 V-ATPase KO 小鼠相似，提示 V-ATPase 功能受损。Western blot 显示 B1 表达减少，B1（V1）和 a4（VO）亚基在 Dmxl1 KO IC 中更多地位于细胞内，且共定位减少。同时，亚细胞分级分离显示 KO 细胞中膜部分的 V1 相关 B1 相对细胞溶胶减少。此外，使用针对 B1 和 a4 V-ATPase 亚基的探针进行的接近连接测定也显示出关联减少。我们提出，Dmxl1 的缺失减少了 V-ATPase 全酶的组装，从而抑制了质子泵的功能。Dmxl1 可能将 V1 结构域募集到膜上，并促进与 VO 结构域的组装，在其缺失的情况下，V1 可能被靶向降解。我们的结论是，Dmxl1 是一种真正的哺乳动物 V-ATPase 组装因子。
