Function of Tubulin Binding Proteins in Vivo

Corresponding author: Frank Solomon, Bldg. E17, Rm. 220, MIT, Cambridge, MA 02139., solomon{at}mit.edu (E-mail)

Communicating editor: M. D. ROSE

Overexpression of the ß-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant -tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests 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 reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-ß-tubulin complex, as it does in the -tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant -tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free ß-tubulin.

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