Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence
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Media type:
E-Article
Title:
Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence
Description:
<jats:title>ABSTRACT</jats:title>
<jats:p>A new member of the tubulin superfamily, γ-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of γ-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa γ-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell super-natant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas γ-tubulin separated into two major peaks. The lagging peak contained a monomeric form of γ-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded γ-tubulin polypeptide from the complex in the presence of MgATP. The released γ-tubulin monomers were capable of binding to microtubules in vitro and biochemical quanti-ties of active monomers were further purified using a com-bination of size-exclusion and ion-exchange column chro-matography. The endogenous Sf9 cell γ-tubulin migrated faster than Chlamydomonas γ-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas γ-tubulin was present in a monomeric form, endogenous γ-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that γ-tubulin could form a heterodimer with hitherto unknown molecule(s).</jats:p>