A Different Conformation for EGC Stator Subcomplex in Solution and in the Assembled Yeast V-ATPase: Possible Implications for Regulatory Disassembly

Abstract

Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

“…All of the features discussed below were apparent at any of these resolutions but most distinctly so at 16 Å, and consequently the figures rendered here are for a map built to 16 Å. The map has a clear handedness and the absolute hand of the map, which was inconsistent between previous published models ( [16][17][18][19][20][21], was found to be correct as represented here by using the Freehand test (41) (Fig. S4B).…”

“…This arrangement is different from the peripheral stalk of the F-type ATP synthase (11,12), which wraps around the long-axis of the complex and the eukaryotic V-ATPase (17,21), where the peripheral stalks have a noticeable curvature. While consistently observed, the role of this bending of the peripheral stalks in some F-and V-type ATPases is not known but could relate to the storage of elastic energy in this part of the assembly.…”

“…Previously determined low-resolution structures of V-ATPases from negatively stained specimens generally share an overall architecture (16)(17)(18)(19)(20). Most eukaryotic enzyme structures show three peripheral stalks with the archaeal and eubacterial structures more clearly showing two peripheral stalks.…”

“…The c-ring in F O is homologous to the c-ring of eukaryotic V-ATPase and the L 12 -ring of the T. thermophilus enzyme (14), whereas the membrane-bound a-subunit contains an additional N-terminal domain in V-ATPase and is called subunit I in the T. thermophilus enzyme. The C-subunit of T. thermophilus forms a part of the central stalk unique to the V-ATPase family, coupling V 1 to the proteolipid-ring of L-subunits (15).Previously determined low-resolution structures of V-ATPases from negatively stained specimens generally share an overall architecture (16)(17)(18)(19)(20). Most eukaryotic enzyme structures show three peripheral stalks with the archaeal and eubacterial structures more clearly showing two peripheral stalks.…”

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…Moreover, according to several recent electron microscopy studies of V-ATPases from different organisms 16,[38][39][40] and small-angle X-ray scattering data of NtpI 1-341 , 26 it was determined that the N-terminal domain of the a-subunit forms an elongated 1-shaped structure. In these studies, it was also suggested that this part of the a-subunit lies parallel to the membrane, adjacent to its cytoplasmic side.…”