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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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Mzt1 is not required for the assembly of the γ-TuSC. The ability of Mzt1 to enhance γ-TuSC complex formation was assessed using proteins produced by an in vitro translation system (IVT). The Alp6 was N-terminally tagged with FLAG-His6, and the rest of the components (Alp4, Gtb1, and Mzt1) were untagged. Samples were prepared as a single IVT reaction in order to produce proteins together in the presence of [S35]methionine. These were subjected to immunoprecipitation using an anti-FLAG antibody. The IP complexes were separated using SDS–PAGE and visualized by autoradiography. Thirty-five percent equivalent of cell extracts used for IP samples were loaded as input and supernatant samples. The ratio of Alp6:Alp4:γ-tubulin in the IP complex did not change in the presence or absence of Mzt1.
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Figure 4: Mzt1 is not required for the assembly of the γ-TuSC. The ability of Mzt1 to enhance γ-TuSC complex formation was assessed using proteins produced by an in vitro translation system (IVT). The Alp6 was N-terminally tagged with FLAG-His6, and the rest of the components (Alp4, Gtb1, and Mzt1) were untagged. Samples were prepared as a single IVT reaction in order to produce proteins together in the presence of [S35]methionine. These were subjected to immunoprecipitation using an anti-FLAG antibody. The IP complexes were separated using SDS–PAGE and visualized by autoradiography. Thirty-five percent equivalent of cell extracts used for IP samples were loaded as input and supernatant samples. The ratio of Alp6:Alp4:γ-tubulin in the IP complex did not change in the presence or absence of Mzt1.

Mentions: The phenotype of the mzt1 shut-off strain is very similar to the phenotypes observed in mutants defective in GCP2Alp4 and GCP3Alp6 (Vardy and Toda, 2000). Because GCP2Alp4 and GCP3Alp6 form γ-TuC together with γ-tubulin Gtb1 (Vardy and Toda, 2000), we examined whether Mzt1 influences γ-TuSC formation. FLAG-tagged GCP3Alp6, nontagged GCP2Alp4, nontagged γ-tubulinGtb1, and nontagged Mzt1 were generated by in vitro translation, and the efficiency of γ-TuSC formation was assessed in the presence and absence of Mzt1. All components were translated together, and an anti-FLAG antibody was used to isolate GCP3Alp6 immunocomplex (Figure 4). In the supernatant fraction, only a trace of GCP3Alp6 was found, indicating that GCP3Alp6 was efficiently captured by anti-FLAG antibody. A substantial proportion of the remaining components were found in the supernatant, indicating that they were present in excess in the reaction to reconstitute γ-TuSC. Although Mzt1 was incorporated into the GCP3Alp6 immunocomplex, it did not affect Alp6-Alp4-Gtb1 complex formation, as seen by comparable protein levels of each component in the complex in the absence or presence of Mzt1. Our result indicates that Mzt1 is unlikely to regulate γ-TuSC formation.


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)

Mzt1 is not required for the assembly of the γ-TuSC. The ability of Mzt1 to enhance γ-TuSC complex formation was assessed using proteins produced by an in vitro translation system (IVT). The Alp6 was N-terminally tagged with FLAG-His6, and the rest of the components (Alp4, Gtb1, and Mzt1) were untagged. Samples were prepared as a single IVT reaction in order to produce proteins together in the presence of [S35]methionine. These were subjected to immunoprecipitation using an anti-FLAG antibody. The IP complexes were separated using SDS–PAGE and visualized by autoradiography. Thirty-five percent equivalent of cell extracts used for IP samples were loaded as input and supernatant samples. The ratio of Alp6:Alp4:γ-tubulin in the IP complex did not change in the presence or absence of Mzt1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814152&req=5

Figure 4: Mzt1 is not required for the assembly of the γ-TuSC. The ability of Mzt1 to enhance γ-TuSC complex formation was assessed using proteins produced by an in vitro translation system (IVT). The Alp6 was N-terminally tagged with FLAG-His6, and the rest of the components (Alp4, Gtb1, and Mzt1) were untagged. Samples were prepared as a single IVT reaction in order to produce proteins together in the presence of [S35]methionine. These were subjected to immunoprecipitation using an anti-FLAG antibody. The IP complexes were separated using SDS–PAGE and visualized by autoradiography. Thirty-five percent equivalent of cell extracts used for IP samples were loaded as input and supernatant samples. The ratio of Alp6:Alp4:γ-tubulin in the IP complex did not change in the presence or absence of Mzt1.
Mentions: The phenotype of the mzt1 shut-off strain is very similar to the phenotypes observed in mutants defective in GCP2Alp4 and GCP3Alp6 (Vardy and Toda, 2000). Because GCP2Alp4 and GCP3Alp6 form γ-TuC together with γ-tubulin Gtb1 (Vardy and Toda, 2000), we examined whether Mzt1 influences γ-TuSC formation. FLAG-tagged GCP3Alp6, nontagged GCP2Alp4, nontagged γ-tubulinGtb1, and nontagged Mzt1 were generated by in vitro translation, and the efficiency of γ-TuSC formation was assessed in the presence and absence of Mzt1. All components were translated together, and an anti-FLAG antibody was used to isolate GCP3Alp6 immunocomplex (Figure 4). In the supernatant fraction, only a trace of GCP3Alp6 was found, indicating that GCP3Alp6 was efficiently captured by anti-FLAG antibody. A substantial proportion of the remaining components were found in the supernatant, indicating that they were present in excess in the reaction to reconstitute γ-TuSC. Although Mzt1 was incorporated into the GCP3Alp6 immunocomplex, it did not affect Alp6-Alp4-Gtb1 complex formation, as seen by comparable protein levels of each component in the complex in the absence or presence of Mzt1. Our result indicates that Mzt1 is unlikely to regulate γ-TuSC formation.

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