CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7

Journal article

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.

Authors: Tan, YZ; Abbas, YM; Wu, JZ; Wu, D; Keon, KA

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Cold Spring Harbor Laboratory Press
• Journal: Life Science Alliance
• Volume: 5
• Issue: 11
• Pages: e202201527-e202201527
• Publication date: 2022-07-06
• DOI: 10.26508/lsa.202201527
• EISSN: 2575-1077
• ISSN: 2575-1077
• Language: English
• Keywords: Biology; Chemistry; Endogeny; Biochemistry; Cell biology; Enzyme; ATPase; Biophysics