05) ( Figures 6A and 6N). Thus, the data indicate that the larger pool of quanta released under these conditions in elp3 mutants stems from a presynaptic defect. To independently test for a presynaptic defect in vesicle release in elp3 mutants, we expressed synaptopHluorin (SpH). SpH is a synaptic vesicle-associated Staurosporine molecular weight pH sensor. At low vesicular pH, SpH GFP is quenched but increases in fluorescence upon vesicle fusion ( Miesenböck et al., 1998). We monitored SpH fluorescence during a 500 ms 100 Hz stimulation paradigm, and while the initial baseline fluorescence (Fo) in controls and elp3 mutants is similar

(data not shown), GFP fluorescence increases to a much higher level in elp3 mutants compared to controls ( Figure 6O). The data indicate that significantly more synaptic vesicles in elp3 mutants fuse during such a bout of stimulation. We do not believe that the increased fluorescence we observe is the result of defects in endocytosis in elp3 mutants, as our analyses have not revealed endocytic defects in the mutants (data not shown), and in addition, a potential Trichostatin A solubility dmso defect in endocytosis would not be expected to significantly

contribute to the increase in fluorescence within this short time period. Given that elp3 mutants show morphological defects at the level of their T bars, we tested whether BRP is a substrate for ELP3-dependent acetylation. First, we expressed Drosophila HIS-ELP3 in E. coli, purified, and refolded the protein ( Figures S6A and S6B). Acetyltransferases are prone to autoacetylation ( Choudhary et al., 2009). We therefore incubated ELP3 with 20 mM Acetyl-CoA for various time periods. Western blots probed with antibodies against acetylated lysine (Ac-K) indicate time-dependent

ELP3 autoacetylation ( Figure 7A). Next, we tested whether our ELP3 protein can acetylate purified histone H3, a well-established target, and tubulin. B3GAT3 Our data indicate both concentration- and time-dependent acetylation of histone H3 ( Figure 7B), but we did not observe ELP3-dependent acetylation of tubulin in wild-type fly lysate, or in lysate prepared from elp3 null mutant animals ( Figures 7C and 7E; data not shown). Thus, although our ELP3 fraction is active, it does not support acetylation of tubulin in vitro. Finally, we tested acetylation of BRP in vitro. We immunoprecipitated BRP from fly heads (see also Figure 8H), incubated these BRP-enriched fractions with Acetyl-CoA and ELP3, and probed western blots with Ac-K ( Figure 7D). As shown in Figures 7D and 7F, we find obvious time-dependent acetylation of BRP. These data indicate that ELP3 is sufficient for the acetylation of the active zone-associated protein BRP. To determine if ELP3 acetylates BRP in vivo, we labeled NMJs with Ac-K. Ac-K labels histones in nuclei (data not shown), microtubules in axons that we marked using the monoclonal antibody Futsch22C10 (Figures 8A and 8B), as well as several features in synaptic boutons (Figures 8A and 8C).