Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain γ‐tubulin

Abstract

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominately in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of α-, β-, and γ-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as γ-tubulin ring complexes. This seems true since both size (estimates, i.e. ∼2MDa) and protein components are similar. Furthermore, both α- and γ-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana γ-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which γ-tubulin complexes contain significant α- and β-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte γ-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of γ-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the γ-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble γ-tubulin complexes in vertebrate ovarian oocytes. Mol. Reprod. Dev. 60: 128–136, 2001.