PDF(Pubmed)
Abstract：
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.
摘要：
质子泵V-ATPase驱动基本的生物过程，例如细胞内细胞器的酸化。严重的,V-ATP酶结构域，V1和VO,必须组装以产生功能性全酶。V-ATPase功能障碍导致癌症，神经变性,糖尿病,以及由分泌质子的肾脏插入细胞（IC）活性降低引起的全身性酸中毒。然而,对哺乳动物中V-ATPase的分子调控知之甚少。我们确定了哺乳动物V-ATPase的一种新的相互作用物，果蝇X染色体基因样1（Dmxl1），又名Rabconnectin-3A。Dmxl1，Rav1p的酵母同源物，是催化域可逆组装的复合物的一部分。我们,因此,假设Dmxl1是哺乳动物V-ATP酶组装因子。这里,我们产生了肾IC特异性Dmxl1敲除（KO）小鼠，尿液酸碱度高，像B1V-ATPaseKO小鼠一样，提示V-ATP酶功能受损。Western印迹显示B1表达减少，B1（V1）和a4（VO）亚基在Dmxl1KOIC中在胞内更多，共定位更少。并行,亚细胞分级分离显示，相对于细胞质，KO细胞膜部分中V1相关的B1较少。此外，a使用针对B1和a4V-ATPase亚基的探针进行的邻近连接测定(PLA)也显示相关性降低。我们建议Dmxl1的丢失减少V-ATPase全酶组装，从而抑制质子泵浦功能。Dmxl1可以将V1结构域募集至膜并促进与VO结构域的组装,并且在其不存在时,V1可以靶向降解。我们得出的结论是Dmxl1是真正的哺乳动物V-ATPase组装因子。
