Function of tubulin binding proteins in vivo.
Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-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-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-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 beta-tubulin.
Fleming
J A
JA
Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Vega
L R
LR
Solomon
F
F
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014714:Vertebrates
C107852:CIN1 protein, S cerevisiae; D002352:Carrier Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; C097366:PAC2 protein, S pombe; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin