Functional Dissection and Hierarchy of Tubulin-folding Cofactor Homologues in Fission Yeast

Abstract

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11^Bcontains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21^Edoes not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11^Binteracts with both α-tubulin and Alp21^E, but not with the cofactor D homologue Alp1, whereas Alp21^Ealso interacts with Alp1^D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11^Bresults in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11^Band the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to α-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21⁺or alp1⁺, whereas alp21deletion is rescued by overexpression ofalp1⁺but notalp11⁺. Finally, the alp1mutant is not complemented by either alp11⁺or alp21⁺. The results suggest that cofactors operate in a linear pathway (Alp11^B-Alp21^E-Alp1^D), each with distinct roles.