γ-Tubulin in mammalian cells: the centrosomal and the cytosolic forms

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Abstract
The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unravelled. The finding of the association with the centrosome of γ-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-γ-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic γ-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total γ-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that γ-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic γ-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of γ-tubulin and Tcp1 with either anti-Tcp1 or anti-γ-tubulin antibodies, that a small part of γ-tubulin participates in Tcp1-γ-tubulin particles. Interestingly, the soluble form of γ-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic γ-tubulin-containing complexes in somatic cells suggests that the overall γ-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.