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. 2020 Aug 10;9:e58157. doi: 10.7554/eLife.58157

Figure 4. Requirement of VASH1-microtubule interactions in α-tubulin detyrosination.

(A,B) Tubulin detyrosination assays of VASH1-SVBP WT or mutants in human cells. HeLa Tet-On cells were co-transfected with Myc-VASH1 WT or mutants and Myc-SVBP WT plasmids. At 24 hr post-transfection, the cells were treated without (A) or with 5 µM Nocodazole (B) for 1 hr. The cell lysates were blotted with the indicated antibodies. Compared with VASH1 WT, VASH1 mutants with multiple glutamate substitutions had slightly slower mobilities. The mobility shift is likely caused by the introduction of multiple negative charges, akin to protein phosphorylation, which also sometimes retards gel mobility. deY-tubulin, detyrosinated α-tubulin. 3E, R234E/R299E/L303E. Experiments were repeated three times with similar results. (C–E) In vitro detyrosination of GMPCPP-stabilized human microtubules (C), the C-terminal peptide of α-tubulin (CTα) fused to GST (D), or free αβ-tubulin heterodimers (E) by the indicated recombinant VASH1–SVBP WT or mutant complexes. Experiments were repeated at least three times with similar results. (F) Coomassie-stained gel of microtubule pelleting assays of VASH1-SVBP WT and mutant complexes. (G) Model of microtubule lattice binding, substrate recognition, and tubulin detyrosination by VASH1-SVBP. The ‘+’ signs indicate positive charges.

Figure 4.

Figure 4—figure supplement 1. Requirement of microtubule-VASH1 interactions in α-tubulin detyrosination.

Figure 4—figure supplement 1.

(A–E) Quantification of the relative detyrosination levels of α-tubulin in Figure 4A (A), 4B (B), 4C (C), 4D (D), and 4E (E). Mean ± s.d.; n = 3 independent experiments. (F) Quantification of the percentage of VASH1-SVBP bound to microtubules in Figure 4F. Mean ± s.d.; n = 3 independent experiments. Significance calculated using two-tailed student’s t-test; between WT and mutants; *p < 0.05, **p < 0.01 ***p < 0.001, and ****p < 0.0001.