Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast.
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(B) contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11(B) interacts with both alpha-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alp1(D). The cellular amount of alpha-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin. Both full-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp1(+), whereas alp21 deletion is rescued by overexpression of 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 pathway (Alp11(B)-Alp21(E)-Alp1(D)), each with distinct roles.
Radcliffe
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PA
Laboratory of Cell Regulation, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
Hirata
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D
Vardy
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Toda
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D000595:Amino Acid Sequence; D016764:Cell Polarity; D003593:Cytoplasm / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D020043:Genes, Essential / Q000235:genetics / Q000502:physiology; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009363:Neoplasm Proteins; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
C110653:Alp1 protein, S pombe; C121736:Alp11 protein, S pombe; C121737:Alp21 protein, S pombe; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; C076744:cytoplasmic linker protein 170; D005998:Glycine