A fourth component of the fission yeast gamma-tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when gamma-tubulin function is compromised.
gamma-Tubulin functions as a multiprotein complex, called the gamma-tubulin complex (gamma-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved gamma-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16(+), as a multicopy suppressor of temperature-sensitive alp6-719 mutants. alp16(+) encodes a 759-amino-acid protein with two conserved regions found in all other members of gamma-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16(+) is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225, alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with gamma-tubulin and cosediments with the gamma-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the gamma-TuC between yeast and higher eukaryotes.
Fujita
Akiko
A
Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, United Kingdom.
Vardy
Leah
L
Garcia
Miguel Angel
MA
Toda
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013831:Thiamine / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
C473565:Alp16 protein, S pombe; C512340:Alp4 protein, S pombe; C512341:Alp6 protein, S pombe; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; D013831:Thiamine