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13:55
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
Published on: April 19, 2013
Subunit interactions at the V1-Vo interface in yeast vacuolar ATPase
Rebecca A Oot1, Stephan Wilkens
1Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
The Journal of Biological Chemistry
|February 28, 2012
Summary
The vacuolar ATPase (V-ATPase) enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Eukaryotic vacuolar ATPase (V-ATPase) regulates cellular processes via reversible dissociation.
- This dissociation involves protein-protein interactions between V(1)-ATPase and V(o)-proton channel domains.
- Previous work identified high-affinity binding of the C subunit's head domain to the EG heterodimer.
Purpose of the Study:
- To characterize the N-terminal domain of the V(o) "a" subunit (a(NT(104-372))).
- To investigate the binding interactions of a(NT(104-372)) with other V-ATPase components.
- To elucidate the molecular mechanisms underlying V-ATPase regulation and dissociation.
Main Methods:
- Generation of a water-soluble a(NT(104-372)) construct.
- Analytical gel filtration chromatography and sedimentation velocity analysis to assess dimerization.
- Small angle X-ray scattering (SAXS) for structural determination.
- Isothermal titration calorimetry (ITC) to quantify binding affinities.
Main Results:
- a(NT(104-372)) exhibits concentration-dependent reversible dimerization.
- A low-resolution molecular envelope of the a(NT(104-372)) dimer was determined via SAXS.
- a(NT(104-372)) binds the C(foot) and EG heterodimer with dissociation constants of 22 μM and 33 μM, respectively.
- These binding sites are spatially close in the intact V-ATPase.
Conclusions:
- The N-terminal domain of the V(o) "a" subunit plays a role in V-ATPase assembly and stability.
- High-avidity interactions involving a(NT(104-372)), C(foot), and EG resist rotational catalysis torque.
- Reversible enzyme dissociation may be initiated by disruption of a(NT(104-372)) interactions with C(foot) or EG.
- An unknown signaling mechanism likely triggers these dissociation events.
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