Structural characterisation of V-ATPases by electron microscopy

Vacuolar H+-translocating ATPases (V-ATPases) are fundamentally important proteins. They pump protons into the interior of most cellular endomembrane compartments at the expense of ATP. V-ATPases are comprised of a membrane-integrated proton-translocating part (V0) and a soluble catalytic part (V1). There are indications for multiple stalks connecting V1 and V0, however up to now three-dimensional information for a complete V-ATPase complex has not been available. <br>The V-ATPase was purified from three sources: lemon fruits, the plant Kalanchoë daigremontiana and the filamentous fungus Neurospora crassa. Structurally and functionally intact V-ATPases were obtained from K. daigremontiana and N. crassa. <br>The architecture of V-ATPase complexes was explored through electron microscopy of single particles and their subsequent digital image processing. Three-dimensional (3D) maps were calculated of the plant V-ATPase – both in the presence and the absence of a non-hydrolysable ATP analogue (AMP-PNP) – and the fungal V-ATPase. <br>In the 3D maps, the details of the three-dimensional organisation of the stalk region between V1 and V0 were revealed for the first time. They show that a central stalk is surrounded by three peripheral stalks of different sizes and shapes. These and previously established results were used to build a model of the subunit arrangement within the V-ATPase complex. Based on this model and the current understanding of the “rotor” mechanism in the closely related F-ATPases, it is possible to explain a similar rotor-stator-machinery in V-ATPases, thereby suggesting that three peripheral stalks form the static bearing. <br>From a regulatory point of view it was found that in the absence of the ATP analogue the V-ATPase was structurally less well defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes might be the initial step in a controlled disassembly upon cellular ATP-depletion.

Author dc:creator

• Domgall, Ines

Contributors dc:contributor

• Gräber, Peter

• V-ATPase

Repository record source_url

https://freidok.uni-freiburg.de/data/648

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:648