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

Abstract

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.

Genetics of continuous colour variation in a pair of sympatric sulphur butterflies

“…Second, homologs of Red contain a LysM domain, and it is their only annotated feature (Francescutti et al, 2022; Grant et al, 2016). The molecular function of this domain is poorly understood in insects, but there is mounting evidence in vertebrates that the LysM domains of several genes interact with the v-ATPase to modulate vacuolar pH across a variety of endosomal organelles (Merkulova et al, 2015; Castroflorio et al, 2021; Eaton et al, 2021; Tan et al, 2022). There is precedent for a role of pterinosome acidification in modulating the red vs. yellow states of squamate pigment cells (Saenko et al, 2013), and the roles of melanosomal pH and v-ATPase activity in fine-tuning melanin content are well established in vertebrates (Ramos-Balderas et al, 2013; Wakamatsu et al, 2021).…”

Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

“…[32] Yeast Oxr1p is a 273-residue globular protein that contains a C-terminal TLDc domain (TLDc stands for Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), Domain catalytic), which has several homologs in mammals, including nuclear receptor coactivator 7 (NCOA7), OXR1, TLDC2, TBC1D24, mammalian enhancer of AKT1-7 (mEAK7), and interferon induced protein 44 (IFI44). [33,34] All of the mammalian TLDc domaincontaining proteins except IFI44 have now been shown to directly interact with V-ATPase, [35][36][37][38][39] and several have also been linked to V-ATPase function. [40,41] In the following sections, we will briefly review the structure and mechanism of the V-ATPase followed by a summary of our current knowledge of the mechanisms of reversible and Oxr1p induced disassembly of the yeast V-ATPase.…”

“…[46] Early studies of the structure of the holoenzyme, its V 1 and V o subcomplexes, as well as individual subunits and subunit complexes were conducted using negative stain electron microscopy, [47][48][49] X-ray crystallography, [50][51][52][53][54] and nuclear magnetic resonance (NMR) spectroscopy. [55] More recent advances in cryo electron microscopy (cryoEM) have provided near-atomic or secondary structure resolution models of the V-ATPases from a variety of organisms, including the enzymes from yeast, [27,32,[56][57][58] rat [59] and bovine brain, [60] pig kidney, [39] human cell culture, [38,61] and lemon. [62] Pioneering cryoEM structural work by the Rubinstein lab resolved populations of purified yeast V-ATPase that showed the central rotor subcomplex halted in three angular positions each 120 • apart, with the three populations referred to as rotary states 1-3.…”

“…[ 36 ] Further, mEAK7 has been identified in V‐ATPase preparations from human cells [ 38,86 ] and pig kidney. [ 39 ] The accompanying cryoEM structures showed the enzymes halted in rotary state 2, with mEAK7 bound to the C‐terminal domains of the non‐catalytic subunit AB pair located behind peripheral stator EG3 (Figure 5B). [ 38,39,86 ] Unlike Oxr1p, mEAK7 has a short C‐terminal α helix that inserts between the catalytic site to contact subunit D. Thus, while both mEAK7 and Oxr1p bind subunit B and a peripheral EG stator, mEAK7 contacts EG3 whereas Oxr1p sits next to EG2 (compare left panel in Figure 5A to views in Figure 5B).…”

“…[ 39 ] The accompanying cryoEM structures showed the enzymes halted in rotary state 2, with mEAK7 bound to the C‐terminal domains of the non‐catalytic subunit AB pair located behind peripheral stator EG3 (Figure 5B). [ 38,39,86 ] Unlike Oxr1p, mEAK7 has a short C‐terminal α helix that inserts between the catalytic site to contact subunit D. Thus, while both mEAK7 and Oxr1p bind subunit B and a peripheral EG stator, mEAK7 contacts EG3 whereas Oxr1p sits next to EG2 (compare left panel in Figure 5A to views in Figure 5B). Moreover, since Oxr1p binds EG2 and subunit B in the state 1 enzyme, its TLDc domain binds the B subunit of the open catalytic site (Figure 5C).…”