Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

Tan, Y. Z.; Abbas, Y. M.; Wu, J. Z.; Wu, D.; Hesketh, G. G.; Bueler, S. A.; Gingras, A.-C.; Robinson, C. V.; Grinstein, S.; Rubinstein, J. L.

doi:10.1101/2021.11.03.466369

bioRxiv · 10.1101/2021.11.03.466369

Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

Tan, Y. Z.·Abbas, Y. M.·Wu, J. Z.·Wu, D.·Hesketh, G. G.·Bueler, S. A.·Gingras, A.-C.·Robinson, C. V.·Grinstein, S.·Rubinstein, J. L.

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Abstract

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~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 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the 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 interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity.

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Tan, Y. Z., Abbas, Y. M., Wu, J. Z., Wu, D., Hesketh, G. G., Bueler, S. A., Gingras, A.-C., Robinson, C. V., Grinstein, S., Rubinstein, J. L.. 2021-11-03. Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound. https://doi.org/10.1101/2021.11.03.466369

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