In the presence of a fragment of the Spc110 adapter, recombinant γTuSC can also oligomerize in vitro ( 10). This is promoted by interaction with adapter proteins at the spindle pole body, a microtubule organizing center (MTOC) that is equivalent to the animal centrosome ( 9). In budding yeast, the nucleation template is formed by γTuSC oligomerization. Higher-order oligomeric assemblies of GCPs function as nucleation templates by presenting γ-tubulin molecules in a circular, helical arrangement that resembles the configuration of α- and β-tubulin in the microtubule ( 3, 4, 6– 8). Other eukaryotes including humans use three additional GCP family members, GCP4, GCP5, and GCP6. These two proteins associate laterally, and each binds one molecule of γ-tubulin, giving rise to a heterotetrameric, “Y”-shaped γ-tubulin small complex (γTuSC) ( 5). Budding yeast contains only two GCPs, GCP2 and GCP3. Nucleation requires another type of tubulin, γ-tubulin, which interacts with members of the conserved gamma complex protein (GCP) family ( 3, 4). Formation, maintenance, and remodeling of these networks crucially depend on microtubule nucleation and its regulation in space and time ( 1, 2).
Networks of microtubules are essential for various cellular functions ranging from chromosome segregation during cell division to intracellular transport. Microtubules are tubular polymers of heterodimers of α- and β-tubulin. Our work finds RUVBL as an assembly factor that regulates γTuRC in cells and allows production of recombinant γTuRC for future in-depth mechanistic studies. We further use cryo-EM to identify features that determine the intricate, higher-order γTuRC architecture. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC as revealed by its cryo–electron microscopy (cryo-EM) structure at ~4.0-Å resolution. RUVBL interacts with γTuRC subcomplexes but is not part of fully assembled γTuRC. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous coexpression system. Here, we show that a complex of RuvB-like protein 1 (RUVBL1) and RUVBL2 “RUVBL” controls assembly and composition of γTuRC in human cells. Since its discovery over two decades ago, γTuRC has evaded in vitro reconstitution and thus detailed structure-function studies. The microtubule nucleator γ-tubulin ring complex (γTuRC) is essential for the function of microtubule organizing centers such as the centrosome.
0 kommentar(er)
