Figure 4.

Sucrose density gradients of pan-neuronally expressed wt and mutant DVMAT. A–F, Postnuclear homogenates from fly heads expressing UAS-DVMAT-wt (A, D), UAS-DVMAT-Δ3 (B, E), or UAS-DVMAT-Y600A (C, F) were separated by sucrose density gradient fractionation. Transgenes were expressed using the elav-Gal4 driver in the dVMAT wt background and were coexpressed with UAS-ANF-GFP. Fractions were probed for DVMAT on Western blots using antibodies to the HA tag common to all DVMAT transgenes, CSP as a marker for SVs, and ANF as a marker for LDCVs, as indicated. A–C show representative Western blots, and D–F show quantitation of immunoblots for HA-DVMAT (solid black line), CSP (dotted gray line), and ANF (dashed gray line) for DVMAT-wt (E) DVMAT-Δ3 (D), and DVMAT-Y600A (F; n = 3–4 gradients per genotype; 2000–4000 flies per gradient). Whereas the major peak of DVMAT-wt is in SV fractions, DVMAT-Y600A shows a bimodal distribution in both light and heavy fractions that partially overlap with markers for SVs and LDCVs. The major peak of DVMAT-Δ3 differs from that of either CSP or ANF-GFP. Note that the scale on the left-hand axes differs for D vs E and F. G, H, The amount of each DVMAT isoform in fractions containing LDCVs (G, fractions 5 and 6) or SVs (H, fractions 10–12) was totaled. Total anti-HA immunoreactivity representing DVMAT differed between genotypes for SV-containing fractions (G, ANOVA, p = 0.01, Dunnett's multiple-comparison test vs wt, *p < 0.05 or **p < 0.01, as indicated) but not LDCV-containing fractions (H).