Limits...
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).

Show MeSH

Related in: MedlinePlus

Confirmation of a direct interaction between Mzt1 and Alp61-186. (A) Purification of recombinant 15N-labeled Mzt1 from bacteria cell extracts. Gel filtration chromatography of 15N-Mzt1-His6 on a Superdex 75 10/300 GL column. Molecular weight markers are indicated by dashed lines. Molecular weights of peaks I, II, and III are estimated to be ∼130, 50, and 35 kDa, respectively. Given that the predicted molecular weight of Mzt1-His6 is expected to be 9.1 kDa, peak I may represent dodecamer, peak II heptamer or hexamer, and peak III tetramer or trimer. SDS–PAGE analysis (4–12% gradient gel) confirmed that all eluted peaks were Mzt1-His6 (arrowhead, molecular weight ∼9 kDa). Peaks II and III were combined, buffer exchanged into NMR buffer (20 mM phosphate, 50 mM NaCl, 2 mM DTT, 10% D2O, pH 6.5), and concentrated for NMR. (B) Purification of recombinant nonlabeled Alp61-186 from bacteria cell extracts. Anion exchange chromatography of Alp61-186 (HiTrap Q HP) using a 10 mM to 1 M sodium chloride gradient (purple line). (C) 1H,15N HSQC spectra of 80 μM 15N-labeled Mzt1 in the absence (blue) and presence (green) of 160 μM Alp61-186. Inset, zoomed-in region showing the progressive chemical shift changes upon addition of 40 (red), 80 (black), and 160 μM (green) Alp61-186. The progressive spectral changes on addition of Alp61-186 are indicative of a direct interaction. (D) CD spectra of Mzt1 alone (green), Alp6 alone (gray), and 1:1 complex of Mzt1:Alp6. The complex trace (pink) shows markedly more secondary structure than the summation of the two isolated proteins (blue). (E) Denaturation profiles for Mzt1 alone (green), Alp6 alone (gray), and a 1:1 complex of Mzt1:Alp6 (pink) were measured by monitoring the change in CD at 222 nm with increasing temperature. Neither Mzt1 nor Alp6 alone showed any cooperative unfolding. However, Mzt1 and Alp6 together showed cooperative unfolding, suggesting that they form a complex. This denaturation profile of the complex was significantly different from the calculated profile of the sum of the two individual components (blue). (F) Schematic diagram of Alp6, highlighting the predicted Mzt1-binding region. Whereas recombinant Mzt1 interacts with Alp61-186, yeast two-hybrid analysis shows that Mzt1 fails to interact with Alp61-117. The Mzt1-binding region of GCP3Alp6 is predicted to include the flexible region linking GD1 and GD2 but not the region including the residues (499–503, indicated in red) corresponding to the proposed “hinge” region of human GCP3 (Guillet et al., 2011).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814152&req=5

Figure 6: Confirmation of a direct interaction between Mzt1 and Alp61-186. (A) Purification of recombinant 15N-labeled Mzt1 from bacteria cell extracts. Gel filtration chromatography of 15N-Mzt1-His6 on a Superdex 75 10/300 GL column. Molecular weight markers are indicated by dashed lines. Molecular weights of peaks I, II, and III are estimated to be ∼130, 50, and 35 kDa, respectively. Given that the predicted molecular weight of Mzt1-His6 is expected to be 9.1 kDa, peak I may represent dodecamer, peak II heptamer or hexamer, and peak III tetramer or trimer. SDS–PAGE analysis (4–12% gradient gel) confirmed that all eluted peaks were Mzt1-His6 (arrowhead, molecular weight ∼9 kDa). Peaks II and III were combined, buffer exchanged into NMR buffer (20 mM phosphate, 50 mM NaCl, 2 mM DTT, 10% D2O, pH 6.5), and concentrated for NMR. (B) Purification of recombinant nonlabeled Alp61-186 from bacteria cell extracts. Anion exchange chromatography of Alp61-186 (HiTrap Q HP) using a 10 mM to 1 M sodium chloride gradient (purple line). (C) 1H,15N HSQC spectra of 80 μM 15N-labeled Mzt1 in the absence (blue) and presence (green) of 160 μM Alp61-186. Inset, zoomed-in region showing the progressive chemical shift changes upon addition of 40 (red), 80 (black), and 160 μM (green) Alp61-186. The progressive spectral changes on addition of Alp61-186 are indicative of a direct interaction. (D) CD spectra of Mzt1 alone (green), Alp6 alone (gray), and 1:1 complex of Mzt1:Alp6. The complex trace (pink) shows markedly more secondary structure than the summation of the two isolated proteins (blue). (E) Denaturation profiles for Mzt1 alone (green), Alp6 alone (gray), and a 1:1 complex of Mzt1:Alp6 (pink) were measured by monitoring the change in CD at 222 nm with increasing temperature. Neither Mzt1 nor Alp6 alone showed any cooperative unfolding. However, Mzt1 and Alp6 together showed cooperative unfolding, suggesting that they form a complex. This denaturation profile of the complex was significantly different from the calculated profile of the sum of the two individual components (blue). (F) Schematic diagram of Alp6, highlighting the predicted Mzt1-binding region. Whereas recombinant Mzt1 interacts with Alp61-186, yeast two-hybrid analysis shows that Mzt1 fails to interact with Alp61-117. The Mzt1-binding region of GCP3Alp6 is predicted to include the flexible region linking GD1 and GD2 but not the region including the residues (499–503, indicated in red) corresponding to the proposed “hinge” region of human GCP3 (Guillet et al., 2011).

Mentions: Purified Mzt1 migrated as a single band on SDS–PAGE with a molecular weight of ∼9 kDa (Figure 6A). However, size exclusion chromatography of purified Mzt1-His6 suggested that Mzt1 exists as several oligomeric species. The major species has an apparent molecular weight of 50 kDa (peak II). Two other species have apparent molecular weights of 130 (peak I) and 35 kDa (peak III), respectively (Figure 6A). In contrast to Mzt1, Alp61-186 purified as a single species (Figure 6B).


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)

Confirmation of a direct interaction between Mzt1 and Alp61-186. (A) Purification of recombinant 15N-labeled Mzt1 from bacteria cell extracts. Gel filtration chromatography of 15N-Mzt1-His6 on a Superdex 75 10/300 GL column. Molecular weight markers are indicated by dashed lines. Molecular weights of peaks I, II, and III are estimated to be ∼130, 50, and 35 kDa, respectively. Given that the predicted molecular weight of Mzt1-His6 is expected to be 9.1 kDa, peak I may represent dodecamer, peak II heptamer or hexamer, and peak III tetramer or trimer. SDS–PAGE analysis (4–12% gradient gel) confirmed that all eluted peaks were Mzt1-His6 (arrowhead, molecular weight ∼9 kDa). Peaks II and III were combined, buffer exchanged into NMR buffer (20 mM phosphate, 50 mM NaCl, 2 mM DTT, 10% D2O, pH 6.5), and concentrated for NMR. (B) Purification of recombinant nonlabeled Alp61-186 from bacteria cell extracts. Anion exchange chromatography of Alp61-186 (HiTrap Q HP) using a 10 mM to 1 M sodium chloride gradient (purple line). (C) 1H,15N HSQC spectra of 80 μM 15N-labeled Mzt1 in the absence (blue) and presence (green) of 160 μM Alp61-186. Inset, zoomed-in region showing the progressive chemical shift changes upon addition of 40 (red), 80 (black), and 160 μM (green) Alp61-186. The progressive spectral changes on addition of Alp61-186 are indicative of a direct interaction. (D) CD spectra of Mzt1 alone (green), Alp6 alone (gray), and 1:1 complex of Mzt1:Alp6. The complex trace (pink) shows markedly more secondary structure than the summation of the two isolated proteins (blue). (E) Denaturation profiles for Mzt1 alone (green), Alp6 alone (gray), and a 1:1 complex of Mzt1:Alp6 (pink) were measured by monitoring the change in CD at 222 nm with increasing temperature. Neither Mzt1 nor Alp6 alone showed any cooperative unfolding. However, Mzt1 and Alp6 together showed cooperative unfolding, suggesting that they form a complex. This denaturation profile of the complex was significantly different from the calculated profile of the sum of the two individual components (blue). (F) Schematic diagram of Alp6, highlighting the predicted Mzt1-binding region. Whereas recombinant Mzt1 interacts with Alp61-186, yeast two-hybrid analysis shows that Mzt1 fails to interact with Alp61-117. The Mzt1-binding region of GCP3Alp6 is predicted to include the flexible region linking GD1 and GD2 but not the region including the residues (499–503, indicated in red) corresponding to the proposed “hinge” region of human GCP3 (Guillet et al., 2011).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Confirmation of a direct interaction between Mzt1 and Alp61-186. (A) Purification of recombinant 15N-labeled Mzt1 from bacteria cell extracts. Gel filtration chromatography of 15N-Mzt1-His6 on a Superdex 75 10/300 GL column. Molecular weight markers are indicated by dashed lines. Molecular weights of peaks I, II, and III are estimated to be ∼130, 50, and 35 kDa, respectively. Given that the predicted molecular weight of Mzt1-His6 is expected to be 9.1 kDa, peak I may represent dodecamer, peak II heptamer or hexamer, and peak III tetramer or trimer. SDS–PAGE analysis (4–12% gradient gel) confirmed that all eluted peaks were Mzt1-His6 (arrowhead, molecular weight ∼9 kDa). Peaks II and III were combined, buffer exchanged into NMR buffer (20 mM phosphate, 50 mM NaCl, 2 mM DTT, 10% D2O, pH 6.5), and concentrated for NMR. (B) Purification of recombinant nonlabeled Alp61-186 from bacteria cell extracts. Anion exchange chromatography of Alp61-186 (HiTrap Q HP) using a 10 mM to 1 M sodium chloride gradient (purple line). (C) 1H,15N HSQC spectra of 80 μM 15N-labeled Mzt1 in the absence (blue) and presence (green) of 160 μM Alp61-186. Inset, zoomed-in region showing the progressive chemical shift changes upon addition of 40 (red), 80 (black), and 160 μM (green) Alp61-186. The progressive spectral changes on addition of Alp61-186 are indicative of a direct interaction. (D) CD spectra of Mzt1 alone (green), Alp6 alone (gray), and 1:1 complex of Mzt1:Alp6. The complex trace (pink) shows markedly more secondary structure than the summation of the two isolated proteins (blue). (E) Denaturation profiles for Mzt1 alone (green), Alp6 alone (gray), and a 1:1 complex of Mzt1:Alp6 (pink) were measured by monitoring the change in CD at 222 nm with increasing temperature. Neither Mzt1 nor Alp6 alone showed any cooperative unfolding. However, Mzt1 and Alp6 together showed cooperative unfolding, suggesting that they form a complex. This denaturation profile of the complex was significantly different from the calculated profile of the sum of the two individual components (blue). (F) Schematic diagram of Alp6, highlighting the predicted Mzt1-binding region. Whereas recombinant Mzt1 interacts with Alp61-186, yeast two-hybrid analysis shows that Mzt1 fails to interact with Alp61-117. The Mzt1-binding region of GCP3Alp6 is predicted to include the flexible region linking GD1 and GD2 but not the region including the residues (499–503, indicated in red) corresponding to the proposed “hinge” region of human GCP3 (Guillet et al., 2011).
Mentions: Purified Mzt1 migrated as a single band on SDS–PAGE with a molecular weight of ∼9 kDa (Figure 6A). However, size exclusion chromatography of purified Mzt1-His6 suggested that Mzt1 exists as several oligomeric species. The major species has an apparent molecular weight of 50 kDa (peak II). Two other species have apparent molecular weights of 130 (peak I) and 35 kDa (peak III), respectively (Figure 6A). In contrast to Mzt1, Alp61-186 purified as a single species (Figure 6B).

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