Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA.
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Simches Research Center, 128 Cambridge St., Boston, MA, 02114, USA.
Sci Rep. 2021 Nov 22;11(1):22654. doi: 10.1038/s41598-021-01809-y.
We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved "TLDc" domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.
我们最近发现核受体共激活因子 7（Ncoa7）和 Oxr1 与质子泵 V-ATPase 相互作用。Ncoa7 和 Oxr1 属于一组在氧化应激反应中发挥作用的蛋白质，它们包含保守的“TLDc”结构域。在这里，我们想知道这个家族的另外三个蛋白质，即 Tbc1d24、Tldc1 和 Tldc2 是否也与 V-ATPase 相互作用，以及 TLDc 结构域是否参与所有这些相互作用。通过共免疫沉淀，内源性肾脏 Tbc1d24（以及 Ncoa7 和 Oxr1）和过表达的 Tldc1 和 Tldc2 均与 V-ATPase 相互作用。此外，纯化的 Ncoa7、O xr1 和 Tldc2 的 TLDc 结构域（但不是 Tbc1d24 或 Tldc1）与 GST 下拉物中的 V-ATPase 相互作用。在氨基酸水平上，保守区域的 Ncoa7 TLDc 结构域中的 G815A、G845A 和 G896A 点突变消除了与 V-ATPase 的相互作用，而 S817A、L926A 和 E938A 突变导致相互作用减少。此外，Ncoa7 和 Tldc2 的 TLDc 结构域上游的多-E 基序与 V-ATPase 的相互作用呈（无显著意义）增强趋势。我们的主要发现是，TLDc 家族的所有五个成员的蛋白质都与 V-ATPase 相互作用。我们的结论是，TLDc 基序定义了一类新的与 V-ATPase 相互作用的调节蛋白。
