IDENTIFICATION OF INTRINSIC DIMER AND OVEREXPRESSED MONOMERIC FORMS OF GAMMA-TUBULIN IN SF9 CELLS INFECTED WITH BACULOVIRUS CONTAINING THE CHLAMYDOMONAS GAMMA-TUBULIN SEQUENCE

A new member of the tubulin superfamily, gamma-tubulin, is localized a t microtubule-organizing centers (MTOCs) in a variety of organisms, Ch lamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expres sion system, Approximately half of the induced 52 kDa gamma-tubulin wa s recovered in the supernatant after centrifugation of Sf9 cell lysate s at 18,000 g for 15 minutes, When the cell supernatant was analyzed b y FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin se parated into two major peaks, The lagging peak contained a monomeric f orm of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The le ading peak, with an apparent molecular mass of 900 kDa, corresponded t o a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. T he released gamma-tubulin monomers were capable of binding to microtub ules in vitro and biochemical quantities of active monomers were furth er purified using a combination of size-exclusion and ion-exchange col umn chromatography, The endogenous Sf9 cell gamma-tubulin migrated fas ter than Chlamydomonas gamma-tubulin with an apparent molecular mass o f 49 kDa on gels, Analyses on gel filtration and sucrose density gradi ent centrifugation showed that, while overexpressed Chlamydomonas gamm a-tubulin was present in a monomeric form, endogenous gamma-tubulin fr om Sf9 and HeLa cells exists as a dimer, These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).