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

Sirajuddin M, Rice LM, Vale RD - Nat. Cell Biol. (2014)

Bottom Line: 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.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.Our results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology and the Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street San Francisco, California 94158, USA.

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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The effects of the α-tubulin C-terminal tyrosine on motor performancea. Microtubules were prepared from tubulin with (TUBA1A/TUBB2A) or without (TUBA1A-ΔY/TUBB2A) the C-terminal tyrosine (Y) on α-tubulin.b. The fold velocity and processivity of de-tyrosinated (ΔY) over tyrosinated (Y) microtubules is shown (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values).c. The effect of the C-terminal α-tubulin tyrosine on microtubule depolymerization by mammalian kinesin-13; the kymographs (time versus microtubule end position) show the decrease in the length of the microtubule polymer over time. Histograms of kinesin-13 microtubule depolymerization rates of tyrosinated (TUBA1A/TUBB2A) and de-tyrosinated (TUBA1A-ΔY/TUBB2A) microtubules. The mean and s.d. were 1.5 ± 0.5 (n = 130) and 0.5 ± 0.2 (n = 92) respectively; n represents the number of microtubule ends analyzed.d. Kinesin-13 depolymerization activity with no α-CTT (Δα), the complete TUBA1A-CTT with its genetically encoded C-terminal tyrosine (α+Y), or the TUBA1A-CTT lacking this tyrosine (αΔY); experiments were performed with a tubulin heterodimer lacking β-CTT (Δβ) or containing the TUBB2A-CTT. Similar results were obtained with the βIV-CTT from two independent experiments (for absolute values and sample number see Table 1).
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Figure 3: The effects of the α-tubulin C-terminal tyrosine on motor performancea. Microtubules were prepared from tubulin with (TUBA1A/TUBB2A) or without (TUBA1A-ΔY/TUBB2A) the C-terminal tyrosine (Y) on α-tubulin.b. The fold velocity and processivity of de-tyrosinated (ΔY) over tyrosinated (Y) microtubules is shown (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values).c. The effect of the C-terminal α-tubulin tyrosine on microtubule depolymerization by mammalian kinesin-13; the kymographs (time versus microtubule end position) show the decrease in the length of the microtubule polymer over time. Histograms of kinesin-13 microtubule depolymerization rates of tyrosinated (TUBA1A/TUBB2A) and de-tyrosinated (TUBA1A-ΔY/TUBB2A) microtubules. The mean and s.d. were 1.5 ± 0.5 (n = 130) and 0.5 ± 0.2 (n = 92) respectively; n represents the number of microtubule ends analyzed.d. Kinesin-13 depolymerization activity with no α-CTT (Δα), the complete TUBA1A-CTT with its genetically encoded C-terminal tyrosine (α+Y), or the TUBA1A-CTT lacking this tyrosine (αΔY); experiments were performed with a tubulin heterodimer lacking β-CTT (Δβ) or containing the TUBB2A-CTT. Similar results were obtained with the βIV-CTT from two independent experiments (for absolute values and sample number see Table 1).

Mentions: We next tested the role of the C-terminal tyrosine in α-tubulin (Fig. 3a), which is subject to a de-tyrosination/tyrosination cycle in metazoans15,16. The presence or absence of the C-terminal tyrosine had little effect on yeast dynein motility (Fig. 3b). When tested on kinesin-1, removal of the C-terminal tyrosine decreased processivity by ~25% (TUBA1A-ΔY compared to TUBA1A; Fig. 3b). Strikingly for kinesin-2, the removal of the tyrosine in α-CTT (TUBA1A-ΔY) caused the opposite effect, increasing velocity and processivity by ~2 and 2.5-fold respectively (Fig. 3b, shown as the ratio of values for ΔY/Y microtubules; absolute values found in Table 1 and Supplementary Table 1). We also tested whether this phenomenon is conserved in kinesin-2 motors from a distant metazoan species. We found that the processivity, but not the velocity, of C. elegans Osm-3 motors could be increased by ~2-fold upon de-tyrosination of α-CTT (Fig. 3b). Our previously described CTT truncation results suggested that α-CTT possesses an inhibitory element for kinesin-2. These results suggest that this inhibitory element is the C-terminal tyrosine.


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

Sirajuddin M, Rice LM, Vale RD - Nat. Cell Biol. (2014)

The effects of the α-tubulin C-terminal tyrosine on motor performancea. Microtubules were prepared from tubulin with (TUBA1A/TUBB2A) or without (TUBA1A-ΔY/TUBB2A) the C-terminal tyrosine (Y) on α-tubulin.b. The fold velocity and processivity of de-tyrosinated (ΔY) over tyrosinated (Y) microtubules is shown (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values).c. The effect of the C-terminal α-tubulin tyrosine on microtubule depolymerization by mammalian kinesin-13; the kymographs (time versus microtubule end position) show the decrease in the length of the microtubule polymer over time. Histograms of kinesin-13 microtubule depolymerization rates of tyrosinated (TUBA1A/TUBB2A) and de-tyrosinated (TUBA1A-ΔY/TUBB2A) microtubules. The mean and s.d. were 1.5 ± 0.5 (n = 130) and 0.5 ± 0.2 (n = 92) respectively; n represents the number of microtubule ends analyzed.d. Kinesin-13 depolymerization activity with no α-CTT (Δα), the complete TUBA1A-CTT with its genetically encoded C-terminal tyrosine (α+Y), or the TUBA1A-CTT lacking this tyrosine (αΔY); experiments were performed with a tubulin heterodimer lacking β-CTT (Δβ) or containing the TUBB2A-CTT. Similar results were obtained with the βIV-CTT from two independent experiments (for absolute values and sample number see Table 1).
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Figure 3: The effects of the α-tubulin C-terminal tyrosine on motor performancea. Microtubules were prepared from tubulin with (TUBA1A/TUBB2A) or without (TUBA1A-ΔY/TUBB2A) the C-terminal tyrosine (Y) on α-tubulin.b. The fold velocity and processivity of de-tyrosinated (ΔY) over tyrosinated (Y) microtubules is shown (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values).c. The effect of the C-terminal α-tubulin tyrosine on microtubule depolymerization by mammalian kinesin-13; the kymographs (time versus microtubule end position) show the decrease in the length of the microtubule polymer over time. Histograms of kinesin-13 microtubule depolymerization rates of tyrosinated (TUBA1A/TUBB2A) and de-tyrosinated (TUBA1A-ΔY/TUBB2A) microtubules. The mean and s.d. were 1.5 ± 0.5 (n = 130) and 0.5 ± 0.2 (n = 92) respectively; n represents the number of microtubule ends analyzed.d. Kinesin-13 depolymerization activity with no α-CTT (Δα), the complete TUBA1A-CTT with its genetically encoded C-terminal tyrosine (α+Y), or the TUBA1A-CTT lacking this tyrosine (αΔY); experiments were performed with a tubulin heterodimer lacking β-CTT (Δβ) or containing the TUBB2A-CTT. Similar results were obtained with the βIV-CTT from two independent experiments (for absolute values and sample number see Table 1).
Mentions: We next tested the role of the C-terminal tyrosine in α-tubulin (Fig. 3a), which is subject to a de-tyrosination/tyrosination cycle in metazoans15,16. The presence or absence of the C-terminal tyrosine had little effect on yeast dynein motility (Fig. 3b). When tested on kinesin-1, removal of the C-terminal tyrosine decreased processivity by ~25% (TUBA1A-ΔY compared to TUBA1A; Fig. 3b). Strikingly for kinesin-2, the removal of the tyrosine in α-CTT (TUBA1A-ΔY) caused the opposite effect, increasing velocity and processivity by ~2 and 2.5-fold respectively (Fig. 3b, shown as the ratio of values for ΔY/Y microtubules; absolute values found in Table 1 and Supplementary Table 1). We also tested whether this phenomenon is conserved in kinesin-2 motors from a distant metazoan species. We found that the processivity, but not the velocity, of C. elegans Osm-3 motors could be increased by ~2-fold upon de-tyrosination of α-CTT (Fig. 3b). Our previously described CTT truncation results suggested that α-CTT possesses an inhibitory element for kinesin-2. These results suggest that this inhibitory element is the C-terminal tyrosine.

Bottom Line: 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.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.Our results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology and the Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street San Francisco, California 94158, USA.

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.

Show MeSH