An α-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.

Microtubule disruption stimulates P-body formation

“…First, we demonstrate that treatment of cells with the drug benomyl stimulates the formation of foci that contain Dcp2p, Dhh1p, and an intermediate of mRNA decay, three constituents of bona fide P-bodies (Sheth and Parker 2003). Second, a genetic lesion in the TUB1 gene (tub1-724) that has been demonstrated to disrupt microtubules under nonpermissive growth conditions (Vega et al 1998) also manifests enhanced formation of P-bodies at the restrictive growth temperature. Finally, we find that disruption of microtubules does not lead to detectable alterations in either mRNA decay or global mRNA translation.…”

“…As an additional means to determine if microtubule destabilization impacts Dcp2p aggregation, we used a strain containing a cold-sensitive allele of TUB1, tub1-724, which results in depletion of polymerized microtubules when grown at the restrictive temperature of 15°C (Vega et al 1998). Wild-type (TUB1) and tub1-724 strains were transformed with plasmid DNA encoding Dcp2p-GFP, and cells were grown at 15°C for the required time prior to monitoring Dcp2p-GFP localization by microscopy (Vega et al 1998). Growth of cells at the restrictive temperature led to the formation of Dcp2p-containing foci in the tub1-724 mutant, while in contrast, Dcp2p foci did not form in wildtype cells (Fig.…”

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

“…To measure Rbl2p/␤-tubulin heterodimer levels directly, we used affinity purification to quantitate the amount of the Rbl2p-His 6 /␤-tubulin complex in cells overexpressing both Rbl2p-His 6 and ␤-tubulin. This method was previously used to identify Rbl2p/␤-tubulin complexes in pGAL-RBL2 tub1-724 cells (33). The mutant ␣-tubulin Tub1-724p binds ␤-tubulin…”

“…Immunoblots against ␣-tubulin and ␤-tubulin were performed as previously described (5,33). Immunoblots against Rbl2p were blocked for Ͼ12 h in 5% nonfat dry milk in TBST (0.5 M Tris, 0.5 M NaCl, 0.1% Tween 20) and incubated with affinity-purified anti-Rbl2p rabbit serum (no.…”

Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

“…Our results uncover a new role of stathmin/Op18 in the integration of signals originating from the MT network itself, thus enabling autoadaptation. Regulation of the steady-state level of polymerized tubulin, which is expected to require an overall negative feedback loop, probably requires many cooperating mechanisms that remain to be discovered but certainly involves capping proteins, MAPs, molecular motors, or factors not participating in the polymerization reaction itself but in other steps such as the formation of the ␣/␤-tubulin dimers (Vega et al, 1998). The positive feedback loop provided by stathmin/Op18 phosphorylation on Ser 16 represents an additional mechanism for MT network control.…”