Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Nat Commun. 2022 Jun 7;13(1):3272. doi: 10.1038/s41467-022-30899-z.
The activity of V-ATPase is well-known to be regulated by reversible dissociation of its V and V domains in response to growth factor stimulation, nutrient sensing, and cellular differentiation. The molecular basis of its regulation by an endogenous modulator without affecting V-ATPase assembly remains unclear. Here, we discover that a lysosome-anchored protein termed (mammalian Enhancer-of-Akt-1-7 (mEAK7)) binds to intact V-ATPase. We determine cryo-EM structure of human mEAK7 in complex with human V-ATPase in native lipid-containing nanodiscs. The structure reveals that the TLDc domain of mEAK7 engages with subunits A, B, and E, while its C-terminal domain binds to subunit D, presumably blocking V-V torque transmission. Our functional studies suggest that mEAK7, which may act as a V-ATPase inhibitor, does not affect the activity of V-ATPase in vitro. However, overexpression of mEAK7 in HCT116 cells that stably express subunit a4 of V-ATPase represses the phosphorylation of ribosomal protein S6. Thus, this finding suggests that mEAK7 potentially links mTOR signaling with V-ATPase activity.
V-ATPase 的活性众所周知可通过其 V 和 V 结构域的可逆解离来调节，这种调节响应于生长因子刺激、营养感应和细胞分化。然而，其受内源性调节剂调节而不影响 V-ATPase 组装的分子基础仍不清楚。在这里，我们发现一种定位于溶酶体的蛋白，称为（哺乳动物 Akt-1-7 增强子（mEAK7）），可与完整的 V-ATPase 结合。我们通过冷冻电镜确定了人源 mEAK7 与天然含脂纳米盘中人源 V-ATPase 的复合物的结构。该结构揭示了 mEAK7 的 TLDc 结构域与亚基 A、B 和 E 结合，而其 C 端结构域与亚基 D 结合，可能阻止 V-V 扭矩传递。我们的功能研究表明，mEAK7 可能作为 V-ATPase 的抑制剂，不会影响体外 V-ATPase 的活性。然而，在稳定表达 V-ATPase 亚基 a4 的 HCT116 细胞中过表达 mEAK7 会抑制核糖体蛋白 S6 的磷酸化。因此，这一发现表明 mEAK7 可能将 mTOR 信号与 V-ATPase 活性联系起来。
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