Abstract
The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.
Highlights
The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms
In a low-resolution cryo-electron microscopy reconstruction of the S. cerevisiae γ-tubulin small complex (γ-TuSC) oligomer, γ-tubulin molecules were observed to be arranged in a left-handed spiral with the same pitch and width as the αβ-tubulin subunits in cellular MTs7,8
We isolated recombinantly expressed C. albicans γ-TuSC from insect cells by affinity purification and anion-exchange chromatography (Supplementary Fig. 1) and used cryo-electron microscopy (cryo-EM) single particle analysis to obtain a structure at near-atomic resolution
Summary
The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. In a low-resolution cryo-electron microscopy (cryo-EM) reconstruction of the S. cerevisiae γ-TuSC oligomer, γ-tubulin molecules were observed to be arranged in a left-handed spiral with the same pitch and width as the αβ-tubulin subunits in cellular MTs7,8 Based on this observation, a model was proposed in which γ-tubulin complexes function as structural templates for MT nucleation. TuSC/CM1 system present in S. cerevisiae is generally comparable to the essential building block of MT nucleation systems in many lower eukaryotes, including the yeast Candida albicans It is to some extent unusual, because the S. cerevisiae genome does not encode for the protein Mozart[1] (Mzt1), which is required for γ-TuSC function in most other organisms[9,10,11,12]. Structural templates for MT nucleation are more versatile in terms of composition and regulation than in
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