Function of tubulin binding proteins in vivo

Overexpression of the beta-tubulin binding protein Rb12p/cofactor A is leth al in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produce s unstable heterodimer. Here we use RBL2 overexpression to identify mutatio ns in other genes that affect formation or stability of heterodimer. This a pproach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heter odimer formation in vivo. The vertebrate homologues of two of these gene pr oducts-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubul in polypeptides into preexisting heterodimer in vitro. Previous work sugges ts that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reac tion. Results presented here demonstrate that these proteins can promote he terodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant ce lls causes microtubule disassembly and enhanced formation of Rb12p-beta-tub ulin complex, as it does in the oc-tubulin mutant that produces weakened he terodimer. Significantly, excess Cin1p/cofactor D suppresses the conditiona l phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential u nder conditions where the levels of dissociated tubulin polypeptides increa se. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of f ree beta-tubulin.