ATPase

Two stators visualized in V-type ATPase

In all organisms ATP serves as a major currency of metabolic energy for driving energy dependent processes. F-type and V-type ATPases are small rotary motors that utilize electrochemical ion gradients (H⁺ or Na⁺) to synthesize ATP, or vice versa. Both types consist of a headpiece, F₁ or V₁, which is connected via a stalk region to the membrane-bound part, F₀ or V₀. A central stalk rotates within a ring of 3 α and 3 ß subunits in F₁, or of 3 A and 3 B in V₁, in discrete steps of 120° . At the F₀/V₀ end the central stalk is connected to a ring of c subunits in the membrane. These subunits are believed to rotate against other subunits of F₀ and V₀, allowing ion translocation at the interface. This implies that additional connection(s), or stator(s), must be present between F₁/V₁ and F₀/V₀, preventing futile rotation of the α3ß3 and A3B3 headpiece. To study the stalk region of the V-type ATPase in detail, we analyzed a set of 7500 single molecular projections of the thermophilic bacterium Caloramator fervidus ATPase by electron microscopy. This work is a collaboration with the group of Konings and Lolkema of the microbiology department from the GBB.

Classification of the projections of detergent-solubilized, negatively stained V₁V₀, reveals a limited number of preferential orientations, in which V₁ is either in a tri-lobed or bi-lobed orientation. The three connections are well separated in the bi-lobed views, as shown in the class-sum of Figure A (left). In the classes with tri-lobed views the left connection is less well resolved, because it is closer to the central stalk and stronger overlapping with V₁ (Figure B, right). The two peripheral connections are each attached to an oval-shaped density on top of V₁. In some particles, one of the oval densities is absent, and this correlates with the loss of a peripheral connection. From these observations we conclude that the intact V-type ATPase has a central stalk and two stator connections.

Possibly, the two stators are unique for V-type ATPases, because in the stalk region of F-type ATPases only the b subunit is considered to be present in two copies, which are thought to form a dimer, participating in one stator. One could ask why the ATPase motor needs such a complicated stator moiety, which seems to have an overall U-shaped form and which must also avoid unwanted friction with the central rotating stalk. The answer is likely related to a higher mechanical stability of the machinery as a whole.