Characterisation of fission yeast alp11 mutants defines three functional domains within tubulin-folding cofactor B

The proper folding of tubulins prior to their incorporation into microtubul es requires a group of conserved proteins called cofactors A to E. In fissi on yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability. Here we show th at the temperature-sensitive alp11-924 mutant, which is defective in the co factor B homologue, contains an opal nonsense mutation, which results in th e production of a truncated Alp11(B) protein (Alp11(1-118)). We isolated a tRNA(Trp) gene as a multicopy suppressor of this mutation, which rescues al p11-924 by read-through of the nonsense codon. The truncated Alp11(1-118) p rotein lacks the C-terminal half of Alp11(B), consisting of a central coile d-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions. Both of these domains are required for t he maintenance of microtubule architecture in vivo. Detailed functional ana lyses lead us to propose that Alp11(B) comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coi l region is necessary for satisfactory maintenance of cellular a-tubulin le vels, and the C-terminal CLIP-170 domain is required for efficient binding to alpha-tubulin.