An alpha-tubulin mutant destabilizes the heterodimer: phenotypic consequences and interactions with tubulin-binding proteins

Abstract

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant alpha-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and beta-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the beta-tubulin-binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind alpha-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.

Figure 1

Synthetic lethal interactions between tub1-724 and altered levels of Rbl2p: a model. Cells expressing tub1-724 as their sole source of α-tubulin die when Rbl2p is either absent or overexpressed. Those relationships are explicable if the heterodimer formed by the Tub1-724p (α*β) dissociates more readily than that formed by the wild-type Tub1p (αβ). In the presence of a normal complement of RBL2, the mutant cells would have a high concentration of free β-tubulin (β_free), which may be responsible for the conditional phenotypes of the mutant (e.g., benomyl supersensitivity). In the absence of Rbl2p, the activity of β_free would increase to toxic levels. In contrast, an excess of Rbl2p could bind to β-tubulin and so enhance dissociation of the mutant heterodimer, promoting dissociation to levels below those necessary for viability.

Figure 2

α- and β-Tubulin coimmunoprecipitate with low efficiency from tub1-724 cells. Immunoblots with anti-α-tubulin (top row) and anti-β-tubulin (bottom row) of whole-cell extracts (WCE) and the precipitates with the two antibodies (αIP and βIP) from wild-type TUB1 or mutant tub1-724 cells.

Figure 3

The Rbl2p-β-tubulin complex in vivo is enhanced in tub1-724 cells. Cells growing in raffinose (0 h) were induced with galactose to express His₆-Rbl2p for 1 or 2 h. His₆-Rbl2p was isolated by affinity chromatography of the whole-cell extracts, and the levels of β-tubulin in the original extract and bound to Rbl2p were assayed by immunoblotting. The results are the averages of two independent experiments for each strain and time point with the ranges indicated by error bars. In both of these experiments, the wild-type strain produced slightly more His₆-Rbl2p upon induction (our unpublished results). Solid bars, TUB1 cells; cross-hatched bars, tub1-724 cells.

Figure 4

Overexpression of RBL2 suppresses TUB1/tub1-724 heterozygous cells. Serial (fourfold) dilutions of saturated cultures were plated to galactose-containing media and allowed to grow at 18°C. The cells were either wild-type diploids or TUB1/tub1-724 cells, carrying either YCpGAL or CEN-GAL-RBL2.

Figure 5

Overexpressing PAC2 is lethal in tub1-724 cells. tub1-724 (triangles) and wild type (squares) containing either control plasmid (open symbols) or GAL-PAC2 (filled symbols) cells growing at 30°C were shifted to galactose-containing media at zero time, and aliquots were taken at intervals and scored for total cells and viable cells.

Figure 6

Microtubule disassembly in tub1-724 cells overexpressing PAC2. Anti-tubulin immunofluorescence of tub1-724 cells containing the control plasmid YCpGAL (A) or a CEN-GAL-PAC2 (B) and wild-type cells containing a CEN-GAL-PAC2 plasmid (C). Cultures were grown in galactose for 3.5 h before fixation for immunofluorescence.

Figure 7

Overexpression of PAC2 is lethal in TUB1/tub1-724 heterozygous cells. Serial (fourfold) dilutions of saturated cultures were plated to galactose-containing media and allowed to grow at 30°C. Strains were wild-type diploids or TUB1/tub1-724 cells containing either YCpGAL or CEN-GAL-PAC2.

Figure 8

Binding of α-tubulin to Pac2p-(HA)-His₆ in double-overexpressing cells. Whole-cell extracts (lanes a, b, e, and f) and eluants from nickel-agarose beads (lanes c, d, g, and h) were analyzed by SDS-PAGE and immunoblotting for HA-tagged Pac2p, α-tubulin, and β-tubulin. The fractions were from cells overexpressing Pac2p-(HA)-His₆ and α-tubulin (a and c), Pac2p-(HA)-His₆ and β-tubulin (e and g), α-tubulin alone (b and d), and β-tubulin alone (d and h). For Pac2p, the bead eluants represent 120 times the load of whole-cell extract. For α- and β-tubulin, the bead eluants represent 500 times the load of whole-cell extract.