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The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro - PubMed

Comparative Study

The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro

Claudia J Bode et al. EMBO Rep. 2003 Jan.

Abstract

The yeast Saccharomyces cerevisiae has two genes for alpha-tubulin, TUB1 and TUB3, and one beta-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents approximately 10% of alpha-tubulin in the cell. We determined the effects of the two alpha-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only alpha-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only alpha-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.

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Figures

Figure 1
Figure 1

SDS–PAGE and isoelectric focusing of yeast tubulin. (A) Coomassie-blue-stained SDS–PAGE gels. Tubulin (2 μg) was loaded onto each lane. Lanes 1–4 contained 99% pure SDS (cat. no. L3771; Sigma-Aldrich, St Louis, MO) and lanes 5 and 6 contained impure SDS (cat. no. L5750; Sigma-Aldrich). Lanes 1 and 5, wild-type tubulin; lane 2, Tub1/Tub2 tubulin; lane 3, Tub3/Tub2 tubulin; lanes 4 and 6, bovine brain tubulin. In lane 1 α1 refers to Tub1 and α2 refers to Tub3. (B) Expanded view of lanes 1–3 from (A). (C) Isoelectric focusing. Lane 1, wild-type tubulin (15 μg); lane 2, Tub1/Tub2 tubulin (15 μg); lane 3, Tub3/Tub2 tubulin (15 μg); lane 4, wild-type (15 μg) and Tub3/Tub2 (7.5 μg) tubulin.

Figure 2
Figure 2

Catastrophic disassembly of representative microtubules. Only the disassembly portions of microtubule lifetimes are shown. Data from individual Tub1/Tub2 microtubules (triangles), wild-type microtubules (circles); and Tub3/Tub2 microtubules (squares) are shown.

Figure 3
Figure 3

Sequence alignment of the two α-tubulin yeast proteins, Tub1 and Tub3. Differences are indicated by the boxes.

Figure 4
Figure 4

Locations of the residues in Tub1 that are different in Tub3. (A) View from the outside of the microtubule. (B) View from the M-loop lateral side of a protofilament. The side chains of amino-acid residues that are different are highlighted in yellow. The lateral elements helix 3 and M-loop are labelled. Orientation axes: + and − represent microtubule orientation; in and out represent the inside and outside of the microtubule, respectively. Structures were drawn as described previously (Gupta et al., 2001).

Figure 5
Figure 5

Residues in Tub1 that are different in Tub3 are located primarily on the outer surface of α-tubulin. The view is from the minus-end of a microtubule. (A) The side chains of the 39 amino-acid residues that are different are highlighted in yellow. (B) The side chains of 12 residues that result in changes of charge and/or involve proline residues are highlighted in red and labelled. Helix 3, the M-loop and the C-terminal region are labelled in (A) and also apply to (B). The C-terminal region is not included in the model.

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