ALPPL2 x Tubulin

The biogenesis of microtubules in the cell comprises a series of complex steps, including protein-folding reactions catalyzed by chaperonins. In addition a group of evolutionarily conserved proteins, called cofactors (A to E), is required for the production of assembly-competent α-/β-tubulin heterodimers. Using fission yeast, in which alp11+, alp1+, and alp21+, encoding the homologs for cofactors B, D, and E, respectively, are essential for cell viability, we have undertaken the genetic analysis of alp31+, the homolog of cofactor A. Gene disruption analysis shows that, unlike the three genes mentioned above, alp31+ is dispensable for cell growth and division. Nonetheless, detailed analysis of alp31-deleted cells demonstrates that Alp31A is required for the maintenance of microtubule structures and, consequently, the proper control of growth polarity. alp31-deleted cells show genetic interactions with mutations in β-tubulin, but not in α-tubulin. Budding yeast cofactor A homolog RBL2 is capable of suppressing the polarity defects of alp31-deleted cells. We conclude that the cofactor-dependent biogenesis of microtubules comprises an essential and a nonessential pathway, both of which are required for microtubule integrity.

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. Alp11Bcontains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21Edoes not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11Binteracts with both α-tubulin and Alp21E, but not with the cofactor D homologue Alp1, whereas Alp21Ealso interacts with Alp1D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11Bresults in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11Band 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 (Alp11B-Alp21E-Alp1D), each with distinct roles.

The main structural components of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of assembly-competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein.