Plant γ-Tubulin Interacts with αβ-Tubulin Dimers and Forms Membrane-Associated Complexes

Abstract

γ-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that γ-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of γ-tubulin with αβ-tubulin dimers. γ-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large γ-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large γ-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate γ-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of γ-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of γ-tubulin suggests additional roles for this protein species in microtubule organization.