Characterization of gamma-tubulin complexes in Aspergillus nidulans and detection of putative gamma-tubulin interacting proteins

Abstract

gamma-Tubulin is central to the nucleation of microtubule assembly in vivo. Although it is most obviously located at microtubule organizing centers, it is also found in soluble cytoplasmic complexes. Characterizing these complexes and identifying proteins that interact with gamma-tubulin in vivo will be necessary if gamma-tubulin function is to be understood fully. We have begun to investigate soluble complexes of gamma-tubulin in Aspergillus nidulans, the organism in which gamma-tubulin was discovered and in which a great deal of genetic and molecular genetic analysis of gamma-tubulin has been carried out. We find that approximately 32% of the gamma-tubulin in A. nidulans is soluble. Sucrose density gradients revealed that the soluble gamma-tubulin is in 8-20S complexes with little or no monomeric gamma-tubulin present. In the presence of 0.5 M KCl the average size of the complexes decreased and a peak was present between 4S and 11S. Cross-linking experiments with a zero-length cross-linker suggest that gamma-tubulin in isolated nuclei and in intact hyphae interacts physically with three proteins with molecular weights of approximately 105, 95, and 80 kDa.