An alpha-tubulin mutant destabilizes the heterodimer: phenotypic consequences and interactions with tubulin-binding proteins.
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.
Vega
L R
LR
Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Fleming
J
J
Solomon
F
F
D003080:Cold Temperature; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006579:Heterozygote; D009154:Mutation; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; C097366:PAC2 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin