Figure 3. Role of the tubulin C terminus and the spastin pore in microtubule-severing.

a, Effects of tubulin C-terminal peptides on microtubule-severing in vitro. Addition of α-Tyr peptide had no detectable effect on severing rates, whereas a β-tubulin C-terminal peptide reduced the severing rate. A scrambled peptide had no detectable effect (see Methods). Scale bar, 5 µm. b, Antibodies (Ab) recognizing Glu–α-tubulin, β-tubulin and polyglutamylated tubulin (anti-‘Glu’) inhibit spastin-mediated severing completely at 1:2 antibody:tubulin molar ratio (the same level of protection was seen even 30 min after spastin addition), whereas antibodies recognizing Tyr– α-tubulin (anti-Tyr) did not protect against severing, even at a 5:1 antibody:tubulin molar ratio (antibody binding to these microtubules demonstrated in Supplementary Fig. 7b). c, Left, conservation of the three pore loops. Loop 1 residues are conserved in all AAA ATPases; loops 2 and 3 are specific to the spastin subfamily. Effects on microtubule-severing of mutations in pore loop residues are shown on top of the alignment: red, inactive; black, severely crippled; green, active. Asterisks denote disease mutations. The effects of mutations generally decrease in severity from the pore entrance to the exit, with loop 1 being the least permissive to substitutions. Right, positions of pore loops (labelled 1, 2 and 3) in the spastin hexamer, in a cross-sectional view of the pore. Side-chains for residues K555, Y556 and D559 as well as residues 592–596 are not visible in the electron density maps and are presumed to be disordered. d, Left, ATPase (red) and microtubule-severing (blue) rates for selected mutants. Error bars represent standard errors of the mean (see Methods). Right, molecular surface of the hexameric spastin model showing in yellow the location of residues on face A that impair severing. Loop residues that impair severing are shown in red.