Fig. 5. Walker B mutant differentially affects the two modes of NSF ATPase activity.

a Preparation of A-MT hybrid NSF that contain ATP-hydrolysis mutant protomer (E329Q). Experimental scheme for A-MT hybrid NSF binding to the SNARE–αSNAP complex. b Comparison of counts of A-MT hybrid NSF and wild-type NSF binding to the SNARE-αSNAP complex under ATP-hydrolyzing (1 mM ATP/10 mM Mg²⁺) and non-hydrolyzing (1 mM ATP/1 mM EDTA) conditions. c Distributions of the deconvoluted photobleaching step(s) for A-MT hybrid NSF at the two conditions in (b) considering 90% labeling efficiency. d, e Measurements of ATP hydrolysis rates without (d) or with (e) SNARE (300 nM) and αSNAP (1 μM) with varying A-MT mutant fractions. f Measured ATP hydrolysis rates in (d, e) normalized to WT samples (0% A-MT). Dashed lines indicate model curves assuming a particular number of mutant subunits per hexamer required to eliminate function. Inset is a magnified view of the dotted box. g Schematic for the removal of WT NSF hexamers in A-MT hybrid NSF samples using magnetic beads. Only NSF containing at least one A-MT subunit stably trapped on the magnetic beads with WT NSF hexamer removed by washing. h Representative SDS-PAGE gel image of proteins binding to magnetic beads under three different conditions after measuring the ATP hydrolysis rate. i Comparison of the measured ATP hydrolysis rate of NSF on the magnetic beads (WT removed) and that of free NSF with 60% A-MT fraction. The two arrows in (d, i) indicate the same data. Error bars in (b) represent mean ± s.d. for n = 10 for A-MT and n = 8 for Wild-type images from two independent experiments. Error bars represent mean ± s.e.m. from three independent experiments (c, e) and four independent experiments (d) and five independent experiments (i). Error bars in (f) represent normalized value ± propagated error by the calculations. The reproducibility of (h) was confirmed in two independent gel images. Source data are provided as a Source Data file.