FIGURE 4.

Tomosyn mutants Δ-loop 1 and Δ-loop 3 display lower binding to SNAP25. PC12 cells expressing EGFP-labeled tomosyn or mutants were immunostained with PLA far-red reagent for identification of interactions with syntaxin and with SNAP25. A, cross-section of cells expressing EGFP-tomosyn. PLA interactions with syntaxin (left) and SNAP25 (right) are marked with purple spots. B, representative projection images of PLA signal between SNAP25 and tomosyn constructs. C, deletion mutants 1 and 3 display significantly lower numbers of PLA signal per cell with SNAP25 compared with the wild type tomosyn (p < 0.01; p < 0.05, respectively). No significant differences were found among tomosyn constructs in PLA signal per cell with syntaxin (p > 0.05). D, analysis showing in vitro GST-syntaxin or GST-SNAP25 pulldown of indicated EGFP-tagged tomosyn proteins. Recombinant GST-syntaxin or GST-SNAP25 was immobilized on glutathione-Sepharose beads and incubated with HEK293A cell lysates expressing EGFP-tomosyn wild type (wt) or the indicated tomosyn loop-deletion construct. Tomosyn immunoreactivity in each fraction was determined using anti-tomosyn antibody. Results show reduced binding of Δ-loops 1 and 3 to GST-SNAP25 compared with tomosyn wild type. No significant differences were found among tomosyn proteins binding to GST-syntaxin. *, p < 0.05; **, p < 0.01.