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Regulation of microtubule motors by tubulin isotypes and post-translational modifications

Abstract

The ‘tubulin-code’ hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin ‘signatures’, which supports the premise of the tubulin-code hypothesis.

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Figure 1: Recombinant tubulin for testing the role of CTTs in motor function.
Figure 2: Minimal CTT requirement for motor function.
Figure 3: The effects of the α-tubulin C-terminal tyrosine on motor performance.
Figure 4: Effects of polyglutamylation on motor performance.
Figure 5: Motility regulation by β-tubulin isotypes.
Figure 6: Cross-regulation of isotype specificity by polyglutamylation.
Figure 7: Summary of CTT-mediated effects on different motors.

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Acknowledgements

The authors thank N. Stuurman for microscopy assistance, M. Tanenbaum for help in lentiviral expression and members of the R.D.V. laboratory for comments on the manuscript. R.D.V. is a Howard Hughes Medical Institute investigator and M.S. is a Human Frontiers Science Program—Long-term fellow (LT-000120/2009). L.M.R. is supported by NSF MCB-1054947. This work received support from an NIH grant (38499).

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M.S. and R.D.V. conceived the project. M.S. performed the experiments, analysed the data and wrote the paper. L.M.R. developed and tested the internal His-tagged tubulin and advised in this study. R.D.V. supervised the work and wrote the paper. All authors discussed and commented on the manuscript.

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Correspondence to Ronald D. Vale.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conservation of microtubule lattice residues.

Sequence conservation of microtubule lattice surface residues of α- (dark grey) and β- (light grey) tubulin. The surface residues are coloured according their conservation between yeast and human tubulin isotypes (red, 100% conservation).

Supplementary Figure 2

Sequence alignment of human α-tubulin isotypes and yeast TUB1, highlighting the conservation of surface residue that are within 6 Å from the kinesin and dynein microtubule binding interfaces, as defined by crystal structure of kinesin-tubulin and cryo-EM structured of dynein-microtubule complexes. This alignment reveals the extremely high degree of conservation of the core compared with the CTTs (see ??a, ??a), with the few cases of substitutions mostly involving similar amino acids.

Supplementary Figure 3

Sequence alignment of human α-tubulin isotypes and yeast TUB2, as indicated in Supplementary Figure 2.

Supplementary Figure 4 Sequence alignment of human α-tubulin isotypes and yeast TUB2, as indicated in Supplementary Figure 2.

a. The site of the His-6 tag in the luminal loop of α-tubulin (light grey) as illustrated. b. Sequence alignment of α-tubulin (yeast TUB1, amino acids 31-68) from different organisms (HS; Human, SS; Sus scrofa, DM; Drosophila melanogaster, SU, Sea Urchin, CE; Caenorhabditis elegans, SC; Saccharomyces cerevisiae). The His6-tag was introduced in between P43 and K44; an insertion of 17 residues in this non-conserved loop does not disrupt tubulin function in yeast60 (see Online Methods). c. SDS-PAGE (left) of the purified recombinant yeast α-int-His/β-tubulin; the DIC image (right) shows microtubules assembled from this tubulin.

Supplementary Figure 5

Sequence alignment of human and Chinese hamster MCAK (kinesin-13) proteins, the motor domain is highlighted in grey. The sequence identity of full length and motor domain are 93% and 96% respectively.

Supplementary Figure 6 Specificity of 10E maleimide towards cysteine at CTTs.

SDS-PAGE of cross-linked products from left to right; Untreated TUBA1A-E542C/TUBB2-E435C; TUBA1A-E542C/TUBB2-E435C + 10E maleimide peptide; untreated TUBA1A/TUBB2 cysteine light mutants (CLM); TUBA1A/TUBB2-CLM + 10E maleimide peptide.

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Supplementary Table 1

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Sirajuddin, M., Rice, L. & Vale, R. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat Cell Biol 16, 335–344 (2014). https://doi.org/10.1038/ncb2920

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