Structure-function relationships in yeast tubulins - PubMed (original) (raw)

Comparative Study

. 2000 May;11(5):1887-903.

doi: 10.1091/mbc.11.5.1887.

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Free PMC article

Comparative Study

Structure-function relationships in yeast tubulins

K L Richards et al. Mol Biol Cell. 2000 May.

Free PMC article

Abstract

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.

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Figures

Figure 1

Figure 1

Phenotypes of TUB1 and TUB2 Ala-scan mutants. The positions of the mutations and their resulting phenotypes are shown for both TUB1 (top) and TUB2 (bottom). The allele number of each mutation is shown, and the altered residues are in bold. SS, benomyl-supersensitive; R, benomyl-resistant; WT, wild-type; CS, cold-sensitive; TS, heat-sensitive; SLOW, slow growth at all temperatures. The TUB2 Ala-scan data is reproduced from Reijo et al. (1994), and phenotype definitions are adapted to be identical to those used in describing the TUB1 mutants.

Figure 2

Figure 2

Growth of tub1 mutants at various temperatures. The growth of each mutant was compared with the growth of the wild-type control across a range of temperatures. Growth ranges from wild-type growth (heavily shaded) to no growth (unshaded).

Figure 3

Figure 3

Nuclear phenotypes and cell cycle progression of tub1 mutants. Large-budded cells were examined both by ascertaining their frequency in a growing population of cells and by staining their nuclei with DAPI. Cells were examined both at 25°C and after a shift to 11°C for two generations. In A, the percentage of large-budded cells with an undivided nucleus was determined; in B, the percentage of large-budded cells with a nucleus away from its normal position near the bud neck was determined; and in C, the percentage of large-budded cells (bud diameter greater than one-half of the mother cell's body) in the population was determined.

Figure 4

Figure 4

Distribution of alanine-scanning mutations in Tub1p. Side chains of amino acids altered by tub1 mutations are colored as in Figure 1: Green, wild-type; red, lethal; blue, cold-sensitive; orange, slow growth; gray, benomyl-supersensitive; purple, benomyl-resistant. Tub2p shown as a backbone trace, and Tub1p is shown as space-filled atoms. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow in Tub2p (Nogales et al., 1999). Orientation axes: + and − show microtubule orientation; H3 and M show lateral sides marked by helix H3 α-helix and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using RASMOL (Sayle and Milner-White, 1995).

Figure 5

Figure 5

Mutations resulting in cold sensitivity are located in areas of the tubulin protein involved in longitudinal or lateral contacts in the microtubule polymer. (A) View from the outside of the microtubule. (B) View from the side of the protofilament. The side chains of amino acids mutated in cold-sensitive alleles (red) and temperature- and cold-sensitive alleles (orange) are shown on Tub2p (top) and Tub1p (bottom) of the yeast tubulin heterodimer. These amino acids are labeled by their allele number. Lateral interaction elements, α-helix H3 and M-loop, are shown in yellow (Nogales et al., 1999). GTP and GDP are shown in green. Adjacent monomers of the protofilament are shown in lighter gray. Orientation axes: + and − show microtubule orientation; latH3 and latM show lateral sides marked by helix H3 and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

Figure 6

Figure 6

Location of benomyl-resistant tub1 and tub2 alleles. (A) View from the outside of the microtubule. (B) View from the side of the protofilament. (C) Cutaway view of the core of β-tubulin, with the interior-facing loop (residues 24–62) removed, β-sheet S1–S6 is shown in orange, and the core helix H7, helix H8, and the intervening T7 loop are shown in red (Nogales et al., 1998b). The side chains of amino acids mutated in alleles resistant to only 40 μg/ml benomyl (red) and alleles resistant to at least 80 μg/ml benomyl (orange) are shown on Tub2p and Tub1p of the yeast tubulin heterodimer, labeled by their allele number. The amino acids mutated in tub2 alleles 431 (E194A, H195A, and E198A), 432 (E194A and D197A), and 433 (E198A) are individually labeled by their residue name and number. The amino acids mutated in tub2 alleles 201, 104, and 150 (Thomas et al., 1985; Machin et al., 1995) are shown in purple. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow (Nogales et al., 1999). GTP and GDP are shown in green. Adjacent monomers of the protofilament are shown in lighter gray. Orientation axes: + and − show microtubule orientation; latH3 and latM show lateral sides marked by helix H3 α-helix and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

Figure 7

Figure 7

Two-hybrid interaction footprint of Bim1p on Tub1p. Bim1p was tested for interaction with each of the Tub1p mutant proteins using the two-hybrid system. Those tub1 mutations that abolished the two-hybrid interaction are shown in red; those tub1 mutations that still interacted are shown in blue (Schwartz et al., 1997). Structure drawn using MIDAS (Ferrin et al., 1988).

Figure 8

Figure 8

tub1 alleles that are synthetically lethal with tub2-201 localize to the intradimer interface. Amino acids mutated in these tub1 alleles (red) are shown on Tub1p (bottom). Tub2p is partly shown above with the tub2-201 mutant residue shown in magenta. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow (Nogales et al., 1999). GTP is shown in green. The view is from the side of the heterodimer. Orientation axes: + and − show microtubule orientation; latH3 and latM show lateral sides marked by helix H3 and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

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