The Mammalian g-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc 97 p and Spc 98 p

g -Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. g -Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic g -tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human g -tubulin were made. The epitope-tagged g -tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic g -tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100and 101-kD components of the g -tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with g -tubulin at the centrosome, cosediment with g -tubulin in sucrose gradients, and coimmunoprecipitate with g -tubulin, indicating that they are part of the g -tubulin complex. The conservation of a complex involving g -tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation. O rganization of the microtubule cytoskeleton occurs through a combination of site-specific nucleation by the centrosome and modulation of microtubule dynamics by interactions with microtubule motors and binding proteins. The centrosome nucleates the assembly of microtubules from soluble tubulin subunits, and maintains an attachment to the minus ends of many of the nucleated microtubules, resulting in a radial array centered at the centrosome. Microtubule motors and binding proteins modify this array, creating specialized structures, such as the mitotic spindle, from the generalized aster. Microtubules act as tracks upon which organelles and vesicles are moved, thus the organization of microtubules is essential to the higher order organization of the cytoplasm. In addition to a and b -tubulin, which make up the microtubule polymer, there exists a third tubulin, g -tubulin, that is localized to the centrosome and not to the microtubule polymer. g -Tubulin was originally identified as a suppressor of a b -tubulin mutation in Aspergillus nidulans (Oakley and Oakley, 1989), and subsequently shown to be conserved in all eukaryotic organisms (Stearns et al., 1991; Zheng et al., 1991; Liu et al., 1994). Although the exact mechanism of microtubule nucleation by the centrosome is not understood, several lines of evidence have implicated g -tubulin as having an essential role in the process. Two approaches to studying g -tubulin have thus far yielded complementary, but largely non-overlapping, information on its function in microtubule organization. Genetic analysis in yeasts and Aspergillus has shown that g -tubulin is essential for viability, is required for mitotic spindle function, and is localized to the spindle pole body (SPB), 1 the fungal equivalent of the centrosome (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991; Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996; Martin et al., 1997). Conditional mutations in the Saccharomyces cerevisiae g -tubulin gene TUB4 result in phenotypes that are consistent with a defect in microtubule nucleation (Marschall et al., 1996; Spang et al., 1996). In the clearest example of such a phenotype, the tub4-34 mutant was found to be able to duplicate the SPB at the restrictive temperature, however one of the duplicated SPBs lacked Address all correspondence to T. Stearns, Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Tel. : (650) 725-6934. Fax: (650) 725-8309. E-mail: stearns@stanford.edu 1. Abbreviations used in this paper : GCP, g -tubulin complex protein; GST, glutathione-S-transferase; SPB, spindle pole body. on S etem er 4, 2017 jcb.rress. org D ow nladed fom