PT  - JOURNAL ARTICLE
AU  - Yong Zi Tan
AU  - Yazan M Abbas
AU  - Jing Ze Wu
AU  - Di Wu
AU  - Kristine A Keon
AU  - Geoffrey G Hesketh
AU  - Stephanie A Bueler
AU  - Anne-Claude Gingras
AU  - Carol V Robinson
AU  - Sergio Grinstein
AU  - John L Rubinstein
TI  - CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7
AID  - 10.26508/lsa.202201527
DP  - 2022 Nov 01
TA  - Life Science Alliance
PG  - e202201527
VI  - 5
IP  - 11
4099  - https://www.life-science-alliance.org/content/5/11/e202201527.short
4100  - https://www.life-science-alliance.org/content/5/11/e202201527.full
SO  - Life Sci. Alliance2022 Nov 01; 5
AB  - 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.