RT Journal Article
SR Electronic
T1 Pump-Rest-Leak-Repeat: regulation of the mammalian-brain V-ATPase via ultra-slow mode-switching
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.06.511076
DO 10.1101/2022.10.06.511076
A1 Eleftherios Kosmidis
A1 Christopher G. Shuttle
A1 Julia Preobraschenski
A1 Marcelo Ganzella
A1 Peter J. Johnson
A1 Salome Veshaguri
A1 Mads P. Møller
A1 Orestis Marantos
A1 Jesper L. Pedersen
A1 Reinhard Jahn
A1 Dimitrios Stamou
YR 2022
UL http://biorxiv.org/content/early/2022/10/07/2022.10.06.511076.abstract
AB Vacuolar-type adenosine triphosphatases (V-ATPases)1–3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyze ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by ~1 V-ATPase molecule per synaptic vesicle6,7. To shed light into this bona fide single-molecule biological process, we investigated electrogenic proton pumping by single mammalian-brain V-ATPases, using individual synaptic vesicles fused with immobilized liposomes. We show V-ATPases do not pump continuously in time, as hypothesized by observing the rotation of bacterial homologs8 and assuming strict ATP/proton coupling. Instead, they stochastically switch between three novel ultra-long-lived proton-pumping, inactive, and proton-leaky modes. Upending conventional wisdom, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. Instead, ATP regulates V-ATPase activity via the switching probability of the proton-pumping mode. In contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. This work reveals and emphasises the mechanistic and biological importance of mode-switching in protein regulation.Competing Interest StatementThe authors have declared no competing interest.