Abstract
Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.
Highlights
Microtubules are nucleated from multiprotein complexes containing ␥-tubulin and associated ␥-tubulin complex proteins (GCPs)
Depletion reduced the recruitment of ␥-tubulin to mitotic centrosomes and to spindle microtubules, which was rescued by siRNA-resistant GCP4 (Fig. 1F)
Using chimeric proteins formed by the N-terminal domain of one GCP fused to the C-terminal domain of another GCP, we show that the N-terminal domains determine the identity of the GCPs
Summary
Microtubules are nucleated from multiprotein complexes containing ␥-tubulin and associated ␥-tubulin complex proteins (GCPs). In most eukaryotes ␥TuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called ␥-tubulin ring complex (␥TuRC). Microtubules are nucleated from specialized multiprotein complexes containing ␥-tubulin and associated proteins [1,2,3] These complexes resemble small rings by electron microscopy and are called ␥-tubulin ring complexes (␥TuRCs) (4 –7). Seven ␥TuSCs assemble stepwise into a helix of one turn plus a small overlap [14] In this helical array, the ␥-tubulin molecules are exposed to form a platform from which ␣/-tubulin dimers assemble into protofilaments. These results led to the prediction that GCPs 4, 5, and 6 integrate into ␥TuRCs in an equivalent manner to GCPs 2 and 3 In this model, all GCPs would interact laterally via their N-terminal domains. This is the first experimental approach supporting that GCPs 4, 5, and 6 are part of the helical wall of the ␥TuRC
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