V-ATPases are rotary nanomachines that pump protons across membranes. Old as the hills, they are found in every eukaryotic cell, diligently pumping protons to acidify intracellular compartments. This enzyme can be viewed as a functional unit of two distinct subcomplexes, the ATP-hydrolyzing V1 and the membrane-bound, proton-conducting V0 complex. There is good evidence that V-ATPase activity is controlled by reversible disassembly of the V1 and V0 complex. Several years ago,a few papers caused a stir by reporting that the V0 complex might have an independent function, because it forms a proteinous pore triggering the fusion of membranes. This is essential for vesicle trafficking, the process that transports different molecules inside cells by encasing them in vesicles that fuse with the target compartments. These findings, however, have not received wide acceptance among cell biologists. Now, an article by Hiesinger and colleagues in a recent issue of Cell may substantially change how we view the V0 complex.
To identify genes that are involved in synaptic malfunction of the Drosophila nervous system, the research team performed an unbiased genetic screen looking for mutant flies that fail to walk towards light. What they found were mutations in the vha100-1 gene, which encodes one brain-specific isoform of the conserved V0 subunit a. Mutations in this gene led to severe defects in neuronal transmission, as indicated by the failure of photoreceptor cells to evoke postsynaptic responses. This suggests that the mutants' neurotransmitter-containing vesicles failed to fuse with the plasma membrane and release the neurotransmitter into the synapse. Since all vesicle fusion was impaired by the mutation, the team concluded that an intact version of the V0 subunit is essential for vesicle fusion. But is the observed decrease in neurotransmission really due to defects in the fusion machinery, or is it caused by the loss of V-ATPase activity, which is necessary to load secretory vesicles with neurotransmitter?
To distinguish between these possibilities, the team took advantage of the fact that yeast cells possess vha100 analogues. They tried to rescue the normal functioning of yeast cells that had lost their vha100 analogues, which showed malfunctions in vacuolar acidification and vesicle trafficking. Interestingly, when the team expressed the Drosophila vha100 protein in the deficient yeast strain, they found that it didn't rescue vacuolar acidification. Instead, it restored vesicle trafficking, suggesting an independent function of vha100 in vesicle fusion.
Experiments with the lipophilic fluorescent dye FM1-43 to examine the effects of the vha100 mutation in Drosophila led the scientists to the same conclusion. They observed decreased fluorescence in the mutants,which indicated fewer vesicle fusions, since the amount of dye uptake is dependent on the number of fusion events. The negative effect on vesicle fusion was obviously independent of vesicle acidification, because a specific V-ATPase inhibitor blocked acidification but had no effect on vesicle fluorescence. In line with this view, Drosophila vha100 mutants had more secretory vesicles in presynaptic photoreceptor cells, where vha100 is enriched in the synaptic terminals. Finally, the team found that vha100 interacts with SNARE proteins, essential components of vesicle fusion machinery.
Hiesinger and colleagues provide the most compelling evidence to date that the V0 complex is involved in vesicle fusion. Their study also bolsters increasing evidence that the complexity of some enzymes is due to their modular composition generating independent functions within a cell.
