Limits...
Mutational analyses reveal a novel function of the nucleotide-binding domain of gamma-tubulin in the regulation of basal body biogenesis.

Shang Y, Tsao CC, Gorovsky MA - J. Cell Biol. (2005)

Bottom Line: These results, coupled with previous studies (Dammermann, A., T.McEwen, G.Khodjakov. 2005.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of gamma-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815-829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713-722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of gamma-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

Show MeSH
γ-Tubulin sequence comparison. Sequence comparison of the sequence of the T3 and T4 loops of γ-tubulin of T. thermophila and five other species with T. thermophila β-tubulin (BTU). GTU, γ-tubuin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171320&req=5

fig2: γ-Tubulin sequence comparison. Sequence comparison of the sequence of the T3 and T4 loops of γ-tubulin of T. thermophila and five other species with T. thermophila β-tubulin (BTU). GTU, γ-tubuin.

Mentions: Previous mutagenesis studies focused on clustered charged residues, which are rare in the NBD, and, therefore, neither study revealed the function of the NBD in γ-tubulin (Hendrickson et al., 2001; Jung et al., 2001). We performed the first mutagenic analysis of the NBD of any γ-tubulin. This domain, conserved in all γ-tubulins, includes loops T1–T6, helix H1, and the NH2-terminal end of helix H7 (Nogales et al., 1998; Nogales, 2001). It shares high sequence homology with the NBD of α/β tubulins (Inclán and Nogales, 2001), and the affinities of γ and β for GTP or guanosine diphosphate (GDP) are not significantly different (Aldaz et al., 2005). However, in addition to the phosphate-binding tubulin signature motif (GGGTGSG) found in the T4 loop of α- and β-tubulins, the NH2-terminal region of the T3 loop in all γ-tubulins contains additional glycine insertions (Fig. 2; Burns, 1995; Inclán and Nogales, 2001; Aldaz et al., 2005) generating a second, similar (GgGAGNN) region, and the crystal structure of human γ-tubulin revealed that unlike β-tubulin, the T3 and T5 loops are disordered in the NBD of γ-tubulin monomers (Aldaz et al., 2005). Thus, the structural differences in the T3 and T5 loops do not affect nucleotide binding, per se, and the γ-tubulin NBD could have unidentified, γ-specific functions in MTOCs.


Mutational analyses reveal a novel function of the nucleotide-binding domain of gamma-tubulin in the regulation of basal body biogenesis.

Shang Y, Tsao CC, Gorovsky MA - J. Cell Biol. (2005)

γ-Tubulin sequence comparison. Sequence comparison of the sequence of the T3 and T4 loops of γ-tubulin of T. thermophila and five other species with T. thermophila β-tubulin (BTU). GTU, γ-tubuin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171320&req=5

fig2: γ-Tubulin sequence comparison. Sequence comparison of the sequence of the T3 and T4 loops of γ-tubulin of T. thermophila and five other species with T. thermophila β-tubulin (BTU). GTU, γ-tubuin.
Mentions: Previous mutagenesis studies focused on clustered charged residues, which are rare in the NBD, and, therefore, neither study revealed the function of the NBD in γ-tubulin (Hendrickson et al., 2001; Jung et al., 2001). We performed the first mutagenic analysis of the NBD of any γ-tubulin. This domain, conserved in all γ-tubulins, includes loops T1–T6, helix H1, and the NH2-terminal end of helix H7 (Nogales et al., 1998; Nogales, 2001). It shares high sequence homology with the NBD of α/β tubulins (Inclán and Nogales, 2001), and the affinities of γ and β for GTP or guanosine diphosphate (GDP) are not significantly different (Aldaz et al., 2005). However, in addition to the phosphate-binding tubulin signature motif (GGGTGSG) found in the T4 loop of α- and β-tubulins, the NH2-terminal region of the T3 loop in all γ-tubulins contains additional glycine insertions (Fig. 2; Burns, 1995; Inclán and Nogales, 2001; Aldaz et al., 2005) generating a second, similar (GgGAGNN) region, and the crystal structure of human γ-tubulin revealed that unlike β-tubulin, the T3 and T5 loops are disordered in the NBD of γ-tubulin monomers (Aldaz et al., 2005). Thus, the structural differences in the T3 and T5 loops do not affect nucleotide binding, per se, and the γ-tubulin NBD could have unidentified, γ-specific functions in MTOCs.

Bottom Line: These results, coupled with previous studies (Dammermann, A., T.McEwen, G.Khodjakov. 2005.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of gamma-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815-829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713-722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of gamma-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

Show MeSH