gammaTubulin at 23C: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - gammaTubulin at 23C

Synonyms - gammaTubulin

Cytological map position - 23C-D

Function - microtubule nucleating factor

Keyword(s) - Cytoskeleton, meiosis, gamma-tubulin ring complex

Symbol - gammaTub23C

FlyBase ID: FBgn0260639

Genetic map position - 2-[6]

Classification - gamma-tubulin

Cellular location - microtubulin organizing center (centriole)



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

In eukaryotic cells, a specialized organelle called the microtubule organizing center (MTOC) is responsible for the disposition of microtubules, helping to form them into a radial, polarized array in interphase cells and in the spindle of mitotic cells. Eukaryotic cells across different species, and different cell types within single species, have morphologically diverse MTOCs, but all these MTOCs share in common the function of organizing microtubule arrays. MTOCs effect microtubule organization by initiating microtubule assembly and anchoring microtubules at their slowly growing minus ends, thus ensuring that in each microtubule array it is the rapidly growing plus ends that extend distally. Gamma-Tubulin is found in the MTOCs of cells from many different organisms, and has several properties that make it a good candidate for both initiation of microtubule assembly and anchorage (Joshi, 1993).

GammaTubulin was first identified as a suppressor of a ß-tubulin mutation in the fungus Aspergillus nidulins. Unexpectedly, antibodies raised against the protein do not stain microtubules, but instead stain the spindle pole body (the MTOC, or centrosome). In Drosophila, gammaTubulin forms a complex with two centrosomal microtubule-associated proteins called CP190 and CP60. Since CP60 can associate with microtubules, and the gammaTubulin-CP190-CP60 complex associates with microtubules, it is believed that the CP60 component binds the complex to microtubules. These observations suggest that gammaTubulin, CP190, and CP60 are all components of a centrosomal complex that can interact with microtubules (Raff, 1993).

CP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. CP60 is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically between late telophase and early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated; dephosphorylated CP60 forms a good substrate for cyclin B/p34cdc2 kinase complexes. Another kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. CP60 binds to purified microtubules; this binding is blocked by CP60 phosphorylation (Kellogg, 1995).

What is the function of gammaTubulin? The microtubule cytoskeleton in animal cells does not assemble spontaneously, but instead requires the function of a centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation. During interphase, the minus (or slow-growing) ends of microtubules are embedded in the PCM and the plus (or fast-growing) ends project outwards into the cytoplasm (or during mitosis, into the spindle apparatus). gammaTubulin is the only component of the PCM thus far implicated in microtubule nucleation. GammaTubulin is localized in the PCM ring structures of purified Drosophila centrosomes. When these centrosomes are used to nucleate microtubule growth, gammaTubulin is localized at the minus ends of the microtubules (Moritz, 1995b).

The gammaTubulin complex has been purified from Xenopus egg extracts. The purified complex is a ring structure with a 25 to 28 nm diameter. Many of these rings appear to be open, with the ends overlapping each other. The wall of a ring is roughly cylindrical: and it is both wider than the wall of a microtubule and higher than an alpha/beta tubulin dimer. The complex nucleates microtubules in vitro, binding to one end of a microtubule. It is concluded that microtubule-nucleating sites within the PCM are ring-shaped templates containing multiple copies of gamma-tubulin. The outer diameter of the complex is similar to the outer diameter of a microtubule. Each complex also has less than two but more than one helical turn, allowing a fit to the end of a microtubule. It is thought that the CP60 and CP190 proteins described above define the framework of a helical structure to which gamma-tubulins bind. The gamma-tubulins then provide a seed for the assembly of alpha/beta tubulin dimers (See Drosophila beta1 Tubulin) that have been stabilized by their interactions with gamma-tubulin. It is thought that the gamma-tubulin complex constitutes the long sought after microtubule nucleating factor in the pericentriolar matrix (Zheng, 1995).


PROTEIN STRUCTURE

Amino Acids - 474

Structural Domains

The Drosophila gammaTubulin shares a 66.7% amino acid identity with A. nidulans gammaTubulin and 78.3 identity with human gammaTubulin (Zheng, 1991).

The exchangeable GTP-binding site on ßtubulin has been extensively studied, but the primary sequence elements that form the binding site on ßtubulin remain unknown. Site-directed mutagenesis of the single ßtubulin gene of S. cerevisiae has been used to test a model for the GTP-binding site on ßtubulin, based on sequence comparisons with members of the GTPase superfamily. The results do not support the proposal that the 203DNEA206 and 295NKAD298 motifs are cognate to motifs found in GTPase superfamily members. Instead, the data argue that the primary sequence elements of tubulins that interact with bound nucleotide, and presumably also those of the alpha- and gamma-tubulin family members, are different from those of "typical" GTPase superfamily members, such as p21ras. The GTPase superfamily should thus be broadened to include not just the typical GTPases that show strong conservation of primary sequence consensus motifs (GxxxxGK, T, DxxG, DxKN) but also "atypical" GTPases, exemplified by the tubulins and other recently identified GTPases, that do not show the consensus motifs of typical GTPases and that also show no obvious primary sequence relationships among themselves. The tubulins and other atypical GTPases thus appear to represent convergent solutions to the GTP-binding and hydrolysis problem (Sage, 1995).


gammaTubulin at 23C: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 30 October 98

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