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

Life Sci Alliance. 2022 Jul 6;5(11):e202201527. doi: 10.26508/lsa.202201527. Print 2022 Nov.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cryoelectron Microscopy
  • Mammals / metabolism
  • Protein Binding
  • Protein Subunits / chemistry
  • Swine
  • Vacuolar Proton-Translocating ATPases* / chemistry
  • Vacuolar Proton-Translocating ATPases* / metabolism


  • Protein Subunits
  • Vacuolar Proton-Translocating ATPases

Associated data

  • PDB/7U8P
  • PDB/7U8Q
  • PDB/7U8R
  • PDB/7U8O