The C-H peripheral stalk base: a novel component in V1-ATPase assembly

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Vacuolar ATPases (V-ATPases) are essential proton pumps that maintain cellular pH gradients.
• They utilize a rotary mechanism driven by ATP hydrolysis, involving a rotor and stator.
• Regulation of V-ATPase activity includes reversible dissociation of its V(1) and V(o) domains, particularly during cellular starvation to conserve ATP.

Purpose of the Study:

• To elucidate the stabilization mechanism of the dissociated V(1)-ATPase.
• To investigate the architecture of peripheral stalks and their binding interactions in the free V(1)-ATPase.
• To understand how rotational catalysis is maintained when the V(1) domain is separated from the membrane-bound V(o) domain.

Main Methods:

• Cryo-electron microscopy was employed to determine the structure of the dissociated V(1)-ATPase.
• Analysis focused on the peripheral stalks and their interactions with other V-ATPase components.

Main Results:

• Confirmed a bridging interaction by subunit F, tethering rotor and stator components in eukaryotic V-ATPases.
• Identified a tight interaction between subunits C and H, forming a base for the three EG peripheral stalks.
• Observed a CE(3)G(3)H sub-assembly, potentially unique to the dissociated V(1)-ATPase, revealing stator architecture.

Conclusions:

• The study clarifies the stabilization mechanism of the free V(1)-ATPase, preventing wasteful ATP hydrolysis.
• The identified CE(3)G(3)H sub-assembly provides insights into the stator architecture of V-ATPases.
• This work suggests a possible intermediate in the assembly of the free V(1)-ATPase.