RT Journal Article
SR Electronic
T1 CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202201527
DO 10.26508/lsa.202201527
VO 5
IS 11
A1 Yong Zi Tan
A1 Yazan M Abbas
A1 Jing Ze Wu
A1 Di Wu
A1 Kristine A Keon
A1 Geoffrey G Hesketh
A1 Stephanie A Bueler
A1 Anne-Claude Gingras
A1 Carol V Robinson
A1 Sergio Grinstein
A1 John L Rubinstein
YR 2022
UL https://www.life-science-alliance.org/content/5/11/e202201527.abstract
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.