Abstract
The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure, than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC.
Highlights
The microtubule (MT)1 cytoskeleton is assembled from highly dynamic polymers of ␣- and -tubulin heterodimers
We report the characterization of a Xenopus gamma ring protein (Xgrip) 210 as a potential subunit for the cap structure that is required for the assembly of the ␥TuRC and its recruitment to the centrosome
To raise rabbit polyclonal antibodies against Xgrip210, a fusion protein was made between glutathione S-transferase (GST) and a fragment of Xgrip210 corresponding to amino acids 874–1097
Summary
The microtubule (MT) cytoskeleton is assembled from highly dynamic polymers of ␣- and -tubulin heterodimers. Most ␥-tubulin in animal cells appears to exist as ␥TuRC (Wiese and Zheng, 1999), and several studies indicated that ␥TuRC is recruited to the centrosome to function as a MT nucleator (Felix et al, 1994; Martin et al, 1998; Moritz et al, 1998; Schnackenberg et al, 1998). The cap structure possibly consists of the non-␥TuSC subunits named Dgrips163 128, and 75s (Keating and Borisy, 2000; Moritz et al, 2000) This structural organization suggests that the cap structure may be important for the assembly of multiple ␥TuSCs into one ␥TuRC. We report the characterization of a Xenopus gamma ring protein (Xgrip) 210 as a potential subunit for the cap structure that is required for the assembly of the ␥TuRC and its recruitment to the centrosome
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