Functional dissection and hierarchy of tubulin-folding cofactor homologuesin fission yeast

We describe the isolation of fission yeast homologues of tubulin-folding co factors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11B contains the glycine-rich motif (t he CLIP-170 domain) involved in microtubular functions, whereas, unlike mam malian cofactor E, Alp21(E) does not. both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis show s that Alp11(B) interacts with both a-tubulin and Alp21(E), but not with th e cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alpl(D). The cellular amount of a-tubulin is decreased in both alp1 and alp(11) muta nts. Overproduction of Alp11(B) results in cell lethality and the disappear ance of microtubules, which is rescued by co-overproduction of alpha-tubuli n. Both hull-length Alp11(B) and the C-terminal third containing the CLIP-1 70 domain localize in the cytoplasm, and this domain is required for effici ent binding to alpha-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp(1+), whereas alp(21) deletion is rescued by overexpression of either alp11(+) or alp21(+). The alp1(+) but not alp11(+). Finally, the alp1 mutant is not complemented by either alp11( +) or alp21(+). The results suggest that cofactors operate in a linear path way (Alp11(B)-Alp21(E)-Alp(D)), each with distinct roles.