Abstract

8 4 5 t e C h n I C a l r e p O r t S Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. The release of neurotransmitters from synaptic vesicles is a key element of chemical synaptic transmission. Synaptic vesicle recycling after exo-and endocytosis requires neurotransmitter uptake by specialized vesicular transporter proteins 1 . The electrochemical driving force for neurotransmitter accumulation is generated by vacuolar-type H + -ATPases (V-ATPases) that actively transport cytosolic protons into the synaptic vesicle lumen, thereby acidifying the vesicles and generating an inside-positive membrane potential 2 . V-ATPases also acidify other subcellular compartments of the secretory and endocytic pathways, such as endosomes, Golgi-derived vesicles and lysosomes, but to a different extent, with lysosomes being the most acidic compartments in a cell (pH < 5) (ref. 3). The tightly regulated acidification of these organelles is a prerequisite for a plethora of different processes, including processing, storage and degradation of proteins, lipids and polysaccharides 3,4 . However, elucidating the physiological roles of V-ATPases has remained challenging because we lack tools that allow rapid and compartment-specific control of proton accumulation. The recent advance of optogenetic methods allows precise manipulation of many cellular activities with light. In neuroscience, microbial rhodopsins such as channelrhodopsins and light-activated ion pumps are applied to modulate the neuronal membrane potential, thereby tuning excitability 5-7 . Cell-type-specific expression of such actuators is commonly achieved by combining sophisticated expression systems with specific promoters 8 , but only few publications report cellcompartment-specific expression of optogenetic actuators, including expression in the postsynaptic density 9 , in dendrites 10 and in axon initial segments RESULTS Targeting Arch3 to synaptic vesicles To functionally express a light-activated proton pump in the synaptic vesicle membrane, we incorporated Arch3 between helix three and four of the vesicular protein synaptophysin, together with the fluorescent proteins mKate2 on the cytosolic and pHluorin on the luminal side to indicate protein expression and localization, as well as luminal acidification. As the C terminus of Arch3 is located on the cytosolic