Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.
Highlights d Basal synaptic transmission in VGLUT1-KO calyx of Held synapses is largely intact d VGLUT1 loss slows down SV refilling with glutamate d VGLUT1 loss leads to abnormal synaptic failures during sustained stimulation d The speed of glutamate loading into SVs can be rate limiting for neurotransmission
The levels and redox states of pyridine nucleotides, such as NADP(H), regulate the cellular redox homeostasis, which is crucial for photooxidative stress response in plants. However, how they are controlled is poorly understood. An Arabidopsis Nudix hydrolase, AtNUDX19, was previously identified to have NADPH hydrolytic activity in vitro, suggesting this enzyme to be a regulator of the NADPH status. We herein examined the physiological role of AtNUDX19 using its loss-of-function mutants. NADPH levels were increased in nudx19 mutants under both normal and high light conditions, while NADP+ and NAD+ levels were decreased. Despite the high redox states of NADP(H), nudx19 mutants exhibited high tolerance to moderate light- or methylviologen-induced photooxidative stresses. This tolerance might be partially attributed to the activation of either or both photosynthesis and the antioxidant system. Furthermore, a microarray analysis suggested the role of ANUDX19 in regulation of the salicylic acid (SA) response in a negative manner. Indeed, nudx19 mutants accumulated SA and showed high sensitivity to the hormone. Our findings demonstrate that ANUDX19 acts as an NADPH pyrophosphohydrolase to modulate cellular levels and redox states of pyridine nucleotides and fine-tunes photooxidative stress response through the regulation of photosynthesis, antioxidant system, and possibly hormonal signaling.
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