Structure of V-ATPase from the mammalian brain

Abstract

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a Legionella pneumophila effector protein. Cryo-electron microscopy allowed the construction of an atomic model, defining the enzyme's ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions.

Fig. 1. Overall structure of brain V-ATPase.

(A) Cycle of synaptic vesicle loading, docking and priming, fusion, and recycling. (B) SDS-PAGE of rat brain V-ATPase isolated with 3×FLAG SidK_1-278 and gel filtration chromatography. (C) Native mass spectrometry of V₁ region (left) and native mass spectrometry of dissociated subunit G (right) at a higher-energy collisional (HCD) voltage of 250 V. The charge state for one peak per subunit is indicated. The table shows the measured mass for each peak (± SD of fit) and the calculated mass depending on subunit composition (Table S5). The difference between calculated and measured masses is indicated. (D) Composite cryoEM map (left) and atomic model (right) of brain V-ATPase in rotational state 1. Scale bar, 25 Å.

Fig. 2. Structure of the V₁ region.

(A) Surface representation. Scale bar, 25 Å. (B) Cross-section through the V₁ region viewed from V₁ towards V_O. ADP is shown in green. (C) Superposition of the catalytic AB pairs (left) and closeup of the conformations of the nucleotide binding sites (right). Scale bar, 5 Å. (D) Interaction of subunits B2, E1, and G2. N and C termini of models are indicated by ‘*’. (E) A continuous β-sheet is formed by E1 and B2 (purple arrowhead).

Fig. 3. Structure of the V_O region.

(A) Surface representation with cryoEM density for subunits f, ATP6AP1/Ac45, and ATP6AP2/PRR. Scale bar, 25 Å. (B) Viewed from V₁ with conserved proton-carrying Glu residues as red spheres. The direction of ATP-hydrolysis-driven rotation of the ring is indicated. (C) Cartoon representation viewed parallel to the plane of the lipid bilayer. (D) Proton path through the V_O region. (E) Surface representation of a1_CTD viewed parallel to the plane of the lipid bilayer. The grey arrow indicates the expected location of an opening between α7 and α8 leading towards the luminal half-channel. (F) Closeup view of the luminal terminus of the luminal half-channel, showing the interaction of subunits f, e2, a1, and the unidentified density. Scale bar, 10 Å.

Fig. 4. Interaction of subunits within the membrane embedded rotor subcomplex.

(A) Subunits ATP6AP1/Ac45, ATP6AP2/PRR, and c″possess transmembrane α-helices in the centre of the c-ring. (B) ATP6AP2/PRR (top) and ATP6AP1/Ac45 (bottom) possess luminal domains that are absent from the structure. SP, signal peptide; Cyt, cytosolic. (C and D) Subunit d1 interacts with the C termini of ATP6AP1/Ac45 (C) and ATP6AP2/PRR (D). (E) Subunits c″, c₍₈₎, and ATP6AP2/PRR interact with the second luminal domain of ATP6AP1/Ac45. Scale bar, 10 Å. (F) Subunits c″, ATP6AP1/Ac45, ATP6AP2/PRR, and d1 create a network of interactions that connect the vesicle lumen and the cytoplasm. Scale bar, 25 Å.