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Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3(Alp6).

Dhani DK, Goult BT, George GM, Rogerson DT, Bitton DA, Miller CJ, Schwabe JW, Tanaka K - Mol. Biol. Cell (2013)

Bottom Line: Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis.Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3).Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

ABSTRACT
In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

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Identification of the Mzt1 initiation methionine. (A) Primary structure of fission yeast Mzt1. CLUSTALW multiple sequence alignment of predicted orthologues of human MOZART1. Fission yeast S. pombe Mzt1 is indicated with a red arrow. Species abbreviations are as follows: Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Dr, Danio rerio; Tn, Tetraodon nigroviridis; Xt, Xenopus tropicalis; Cn, Cryptococcus neoformans; Ci, Ciona intestinalis; Sp, S. pombe; Gl, Giardia lamblia; At, Arabidopsis thaliana; Ol, Ostreococcus lucimarinus; Pt, Paramecium tetraurelia; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster. (B) Two potential initiation methionines in the predicted Mzt1 ORF. Methionine in blue is the initiation methionine for the long Mzt1 sequence (mzt1-L) predicted by Hutchins et al. (2010). Methionine in red is the physiological initiation methionine for the short Mzt1 sequence (mzt1-S). (C) Schematic of the strain (KT3519 and KT3522) used to determine the initiation methionine of Mzt1. The mzt1 gene is tagged with GFP-2xFLAG at its endogenous locus, which is located on chromosome I. The mzt1-S or mzt1-L tagged with GFP-2×FLAG was integrated at the leu1+ locus (chromosome II) under the inducible nmt81 promoter. (D) The yeast strains KT3519 and KT3522, harboring mzt1-S or -L tagged with GFP-2xFLAG at the leu1+ gene locus, under the nmt81 promoter and mzt1-GFP-2xFLAG at its endogenous locus, were grown in the absence (–) or presence (+) of thiamine, which acts to repress the nmt81 promoter. Denatured whole-cell extracts were prepared from these cells, which were subjected to Western blotting. Monoclonal anti-FLAG antibody was used to probe for GFP-tagged Mzt1. α-Tubulin was used as a reference for loading. The blot shows that in the noninduced lanes (–), the bands present correspond to the Mzt1-S.
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Figure 1: Identification of the Mzt1 initiation methionine. (A) Primary structure of fission yeast Mzt1. CLUSTALW multiple sequence alignment of predicted orthologues of human MOZART1. Fission yeast S. pombe Mzt1 is indicated with a red arrow. Species abbreviations are as follows: Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Dr, Danio rerio; Tn, Tetraodon nigroviridis; Xt, Xenopus tropicalis; Cn, Cryptococcus neoformans; Ci, Ciona intestinalis; Sp, S. pombe; Gl, Giardia lamblia; At, Arabidopsis thaliana; Ol, Ostreococcus lucimarinus; Pt, Paramecium tetraurelia; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster. (B) Two potential initiation methionines in the predicted Mzt1 ORF. Methionine in blue is the initiation methionine for the long Mzt1 sequence (mzt1-L) predicted by Hutchins et al. (2010). Methionine in red is the physiological initiation methionine for the short Mzt1 sequence (mzt1-S). (C) Schematic of the strain (KT3519 and KT3522) used to determine the initiation methionine of Mzt1. The mzt1 gene is tagged with GFP-2xFLAG at its endogenous locus, which is located on chromosome I. The mzt1-S or mzt1-L tagged with GFP-2×FLAG was integrated at the leu1+ locus (chromosome II) under the inducible nmt81 promoter. (D) The yeast strains KT3519 and KT3522, harboring mzt1-S or -L tagged with GFP-2xFLAG at the leu1+ gene locus, under the nmt81 promoter and mzt1-GFP-2xFLAG at its endogenous locus, were grown in the absence (–) or presence (+) of thiamine, which acts to repress the nmt81 promoter. Denatured whole-cell extracts were prepared from these cells, which were subjected to Western blotting. Monoclonal anti-FLAG antibody was used to probe for GFP-tagged Mzt1. α-Tubulin was used as a reference for loading. The blot shows that in the noninduced lanes (–), the bands present correspond to the Mzt1-S.

Mentions: The previous study of Mzt1 protein predicted a longer, 97–amino acid, protein (Hutchins et al., 2010). The initiator methionine predicted by us corresponds to the methionine at position 34 of the Mzt1 protein predicted in their study, which we refer to as Mzt1-L. We noticed that the first 33 amino acids of Mzt1-L do not show substantial homology to MOZART1 homologues from a range of organisms (Figure 1A). This prompted us to examine which methionine is used as the start codon.


Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3(Alp6).

Dhani DK, Goult BT, George GM, Rogerson DT, Bitton DA, Miller CJ, Schwabe JW, Tanaka K - Mol. Biol. Cell (2013)

Identification of the Mzt1 initiation methionine. (A) Primary structure of fission yeast Mzt1. CLUSTALW multiple sequence alignment of predicted orthologues of human MOZART1. Fission yeast S. pombe Mzt1 is indicated with a red arrow. Species abbreviations are as follows: Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Dr, Danio rerio; Tn, Tetraodon nigroviridis; Xt, Xenopus tropicalis; Cn, Cryptococcus neoformans; Ci, Ciona intestinalis; Sp, S. pombe; Gl, Giardia lamblia; At, Arabidopsis thaliana; Ol, Ostreococcus lucimarinus; Pt, Paramecium tetraurelia; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster. (B) Two potential initiation methionines in the predicted Mzt1 ORF. Methionine in blue is the initiation methionine for the long Mzt1 sequence (mzt1-L) predicted by Hutchins et al. (2010). Methionine in red is the physiological initiation methionine for the short Mzt1 sequence (mzt1-S). (C) Schematic of the strain (KT3519 and KT3522) used to determine the initiation methionine of Mzt1. The mzt1 gene is tagged with GFP-2xFLAG at its endogenous locus, which is located on chromosome I. The mzt1-S or mzt1-L tagged with GFP-2×FLAG was integrated at the leu1+ locus (chromosome II) under the inducible nmt81 promoter. (D) The yeast strains KT3519 and KT3522, harboring mzt1-S or -L tagged with GFP-2xFLAG at the leu1+ gene locus, under the nmt81 promoter and mzt1-GFP-2xFLAG at its endogenous locus, were grown in the absence (–) or presence (+) of thiamine, which acts to repress the nmt81 promoter. Denatured whole-cell extracts were prepared from these cells, which were subjected to Western blotting. Monoclonal anti-FLAG antibody was used to probe for GFP-tagged Mzt1. α-Tubulin was used as a reference for loading. The blot shows that in the noninduced lanes (–), the bands present correspond to the Mzt1-S.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814152&req=5

Figure 1: Identification of the Mzt1 initiation methionine. (A) Primary structure of fission yeast Mzt1. CLUSTALW multiple sequence alignment of predicted orthologues of human MOZART1. Fission yeast S. pombe Mzt1 is indicated with a red arrow. Species abbreviations are as follows: Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Dr, Danio rerio; Tn, Tetraodon nigroviridis; Xt, Xenopus tropicalis; Cn, Cryptococcus neoformans; Ci, Ciona intestinalis; Sp, S. pombe; Gl, Giardia lamblia; At, Arabidopsis thaliana; Ol, Ostreococcus lucimarinus; Pt, Paramecium tetraurelia; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster. (B) Two potential initiation methionines in the predicted Mzt1 ORF. Methionine in blue is the initiation methionine for the long Mzt1 sequence (mzt1-L) predicted by Hutchins et al. (2010). Methionine in red is the physiological initiation methionine for the short Mzt1 sequence (mzt1-S). (C) Schematic of the strain (KT3519 and KT3522) used to determine the initiation methionine of Mzt1. The mzt1 gene is tagged with GFP-2xFLAG at its endogenous locus, which is located on chromosome I. The mzt1-S or mzt1-L tagged with GFP-2×FLAG was integrated at the leu1+ locus (chromosome II) under the inducible nmt81 promoter. (D) The yeast strains KT3519 and KT3522, harboring mzt1-S or -L tagged with GFP-2xFLAG at the leu1+ gene locus, under the nmt81 promoter and mzt1-GFP-2xFLAG at its endogenous locus, were grown in the absence (–) or presence (+) of thiamine, which acts to repress the nmt81 promoter. Denatured whole-cell extracts were prepared from these cells, which were subjected to Western blotting. Monoclonal anti-FLAG antibody was used to probe for GFP-tagged Mzt1. α-Tubulin was used as a reference for loading. The blot shows that in the noninduced lanes (–), the bands present correspond to the Mzt1-S.
Mentions: The previous study of Mzt1 protein predicted a longer, 97–amino acid, protein (Hutchins et al., 2010). The initiator methionine predicted by us corresponds to the methionine at position 34 of the Mzt1 protein predicted in their study, which we refer to as Mzt1-L. We noticed that the first 33 amino acids of Mzt1-L do not show substantial homology to MOZART1 homologues from a range of organisms (Figure 1A). This prompted us to examine which methionine is used as the start codon.

Bottom Line: Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis.Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3).Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

ABSTRACT
In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

Show MeSH
Related in: MedlinePlus