The Role of a Neuron-Specific V-Atpase in Synapse Specification, Function, and Maintenance

The Vacuolar-type (V-) adenosine triphosphatase (ATPase) is a proton-pumping nanomachine consisting of two multi-subunit, reversibly associating protein sectors, the cytosolic V1 sector and the membrane-bound Vo sector. The V1/Vo holoenzyme hydrolyses ATP to translocate protons across biological membranes thereby modulating lumenal and extracellular pH. Additionally, accumulating evidence suggests that the Vo sector has a role in membrane fusion when dissociated from the V1 sector, its proton-pumping partner. Published evidence for this includes a null allele for the neuron-specific a subunit in the Drosophila Vo sector, v100, which leads to defects in synaptic function that are unrelated to pH regulation. My project emerged from the need to explain why v100 has two additional phenotypes that are absent in other synaptic function mutants: functional and structural degeneration in photoreceptor cells and patterning defects in the visual system neuropil. I proposed that v100 has a previously undocumented role on a neuron-specific endo/lysosomal pathway in addition to its documented role in neurotransmitter secretion.

To test my hypothesis in the context of only one of the two purported v100 functions, I generated transgenic animals with v100 mutations designed to specifically disrupt either acidification or membrane fusion. Using these genetic tools, I discovered that v100 has an essential role in sorting cargo into an endo-lysosomal pathway that concomitantly requires v100 for the acidification-dependent maturation of degradation-competent organelles. This 'sort-and-degrade' mechanism for v100 defines a neuron-specific degradation pathway that is required for synaptic specification, function, and maintenance. In developmental stages, v100 is required to 'sort-and-degrade' guidance receptors as part of the synapse specification program. In the adult, the 'sort-and-degrade' mechanism provides additional degradative capacity to neurons, a cell type that must often maintain homeostasis for unusually long periods of time. Finally, I provide evidence that the role for V100 in membrane fusion requires a direct, physical interaction with Syntaxin-1, an interaction that can be specifically disrupted in vitro and in vivo.

In brief, my results provide mechanistic insight into the acidification-independent role of v100 and reveal the existence of a neuron-specific endo-lysosomal pathway on which v100 functions to 'sort-and-degrade' cargo in order to meet the special needs of a neuron in development, function, and maintenance. [Keywords: Drosophila, intracellular trafficking, development, degeneration, lysosomal, endosomal, degradation, acidification]