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 microtubules requires a group of conserved proteins called cofactors A to E. In fission 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 that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11B protein (Alp11(1-118). We isolated a tRNA(Trp) gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon. The truncated Alp1-118 protein lacks the C-terminal half of Alp11B, consisting of a central coiled-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 the maintenance of microtubule architecture in vivo. Detailed functional analyses lead us to propose that Alp11B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular alpha-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to alpha-tubulin.
Radcliffe
P A
PA
Laboratory of Cell Regulation, Imperial Cancer Research Fund, London, UK.
Toda
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D000595:Amino Acid Sequence; D001483:Base Sequence; D018833:Chaperonins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012364:RNA, Transfer, Trp / Q000737:chemistry / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D029702:Schizosaccharomyces pombe Proteins; D013489:Suppression, Genetic; D013696:Temperature; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
C121736:Alp11 protein, S pombe; D005656:Fungal Proteins; D012331:RNA, Fungal; D012364:RNA, Transfer, Trp; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; D018833:Chaperonins