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The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane.

Chen J, Smoyer CJ, Slaughter BD, Unruh JR, Jaspersen SL - J. Cell Biol. (2014)

Bottom Line: We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication.Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component.Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.

ABSTRACT
In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain-containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1-Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

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Mps3 binding to Ndc1 on the NE is antagonized by Pom152. (A) Prey plasmids producing the indicated protein (Nbp1, Mps3, or Pom34) and bait plasmids containing NDC1 or ndc1-L562S were introduced into wild-type cells used for MYTH (SLJ5572) or a version containing pom152Δ (SLJ6066). 10-fold serial dilutions of cells were spotted onto SD-Leu-Trp and SD-Leu-Trp-His-Ade and plates were incubated for 2 d at 30°C. (B) Schematic demonstrating the principle of FCCS. Simulated data and subsequent correlation curves for co-diffusing red and green particles (top) and for randomly diffusing particles (bottom) is shown. The amplitude of the cross-correlation relative to the auto-correlation curves is indicative of the strength of the interaction. (C) A line profile was generated spanning the NE and line-scanning FCCS data were collected in cells containing Ndc1-mTurquoise2 (green) and Mps3-YFP (red). Bar, 2 µm. (D) Each scan along the line can be visualized in a kymograph, which shows at each time point the fluctuations in molecules and complexes as they traverse the NE. Bar, 2 µm. Correlation analysis of intensity fluctuations was performed at the sites where the line-scan crossed the NE. (E) Average auto-correlation and cross-correlation curves and fits are shown for Ndc1-mTurquoise2 and Mps3-YFP in wild-type (SLJ7436) and pom152Δ (SLJ7438) cells. Error bars represent the SEM. Inset, magnification of plots to show differences in cross-correlation between strains. (F) The apparent fraction Mps3 and Ndc1 bound is increased in pom152Δ cells (P < 0.0001). Error bars represent Monte Carlo standard errors. (G and H) FCCS was also analyzed in wild-type (SLJ7835) and pom152Δ (SLJ7836) strains containing Ndc1-mTurquoise (green) and Nup49-YFP (red), and analyzed as in E and F. In H, the bound fraction presumably reflects the NPC.
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fig6: Mps3 binding to Ndc1 on the NE is antagonized by Pom152. (A) Prey plasmids producing the indicated protein (Nbp1, Mps3, or Pom34) and bait plasmids containing NDC1 or ndc1-L562S were introduced into wild-type cells used for MYTH (SLJ5572) or a version containing pom152Δ (SLJ6066). 10-fold serial dilutions of cells were spotted onto SD-Leu-Trp and SD-Leu-Trp-His-Ade and plates were incubated for 2 d at 30°C. (B) Schematic demonstrating the principle of FCCS. Simulated data and subsequent correlation curves for co-diffusing red and green particles (top) and for randomly diffusing particles (bottom) is shown. The amplitude of the cross-correlation relative to the auto-correlation curves is indicative of the strength of the interaction. (C) A line profile was generated spanning the NE and line-scanning FCCS data were collected in cells containing Ndc1-mTurquoise2 (green) and Mps3-YFP (red). Bar, 2 µm. (D) Each scan along the line can be visualized in a kymograph, which shows at each time point the fluctuations in molecules and complexes as they traverse the NE. Bar, 2 µm. Correlation analysis of intensity fluctuations was performed at the sites where the line-scan crossed the NE. (E) Average auto-correlation and cross-correlation curves and fits are shown for Ndc1-mTurquoise2 and Mps3-YFP in wild-type (SLJ7436) and pom152Δ (SLJ7438) cells. Error bars represent the SEM. Inset, magnification of plots to show differences in cross-correlation between strains. (F) The apparent fraction Mps3 and Ndc1 bound is increased in pom152Δ cells (P < 0.0001). Error bars represent Monte Carlo standard errors. (G and H) FCCS was also analyzed in wild-type (SLJ7835) and pom152Δ (SLJ7836) strains containing Ndc1-mTurquoise (green) and Nup49-YFP (red), and analyzed as in E and F. In H, the bound fraction presumably reflects the NPC.

Mentions: Using the MYTH system, we compared binding of Ndc1 and ndc1-L562S to Pom34, Mps3, and Nbp1 in the presence and absence of POM152 to better understand how Ndc1 is distributed between NE complexes. Deletion of POM152 did not affect binding of Pom34 or Nbp1 to either wild-type or the mutant versions of Ndc1 that we tested (Fig. 6 A). Pom152 was also not required for Ndc1 interaction with other proteins such as Yop1 and Nup59. (Fig. S3 B). However, pom152Δ significantly enhanced association of ndc1-L562S and Mps3 as well as increased binding of Ndc1 and Mps3 (Fig. 6 A), indicating that Pom152 and Mps3 likely act antagonistically to control the distribution of Ndc1.


The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane.

Chen J, Smoyer CJ, Slaughter BD, Unruh JR, Jaspersen SL - J. Cell Biol. (2014)

Mps3 binding to Ndc1 on the NE is antagonized by Pom152. (A) Prey plasmids producing the indicated protein (Nbp1, Mps3, or Pom34) and bait plasmids containing NDC1 or ndc1-L562S were introduced into wild-type cells used for MYTH (SLJ5572) or a version containing pom152Δ (SLJ6066). 10-fold serial dilutions of cells were spotted onto SD-Leu-Trp and SD-Leu-Trp-His-Ade and plates were incubated for 2 d at 30°C. (B) Schematic demonstrating the principle of FCCS. Simulated data and subsequent correlation curves for co-diffusing red and green particles (top) and for randomly diffusing particles (bottom) is shown. The amplitude of the cross-correlation relative to the auto-correlation curves is indicative of the strength of the interaction. (C) A line profile was generated spanning the NE and line-scanning FCCS data were collected in cells containing Ndc1-mTurquoise2 (green) and Mps3-YFP (red). Bar, 2 µm. (D) Each scan along the line can be visualized in a kymograph, which shows at each time point the fluctuations in molecules and complexes as they traverse the NE. Bar, 2 µm. Correlation analysis of intensity fluctuations was performed at the sites where the line-scan crossed the NE. (E) Average auto-correlation and cross-correlation curves and fits are shown for Ndc1-mTurquoise2 and Mps3-YFP in wild-type (SLJ7436) and pom152Δ (SLJ7438) cells. Error bars represent the SEM. Inset, magnification of plots to show differences in cross-correlation between strains. (F) The apparent fraction Mps3 and Ndc1 bound is increased in pom152Δ cells (P < 0.0001). Error bars represent Monte Carlo standard errors. (G and H) FCCS was also analyzed in wild-type (SLJ7835) and pom152Δ (SLJ7836) strains containing Ndc1-mTurquoise (green) and Nup49-YFP (red), and analyzed as in E and F. In H, the bound fraction presumably reflects the NPC.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926959&req=5

fig6: Mps3 binding to Ndc1 on the NE is antagonized by Pom152. (A) Prey plasmids producing the indicated protein (Nbp1, Mps3, or Pom34) and bait plasmids containing NDC1 or ndc1-L562S were introduced into wild-type cells used for MYTH (SLJ5572) or a version containing pom152Δ (SLJ6066). 10-fold serial dilutions of cells were spotted onto SD-Leu-Trp and SD-Leu-Trp-His-Ade and plates were incubated for 2 d at 30°C. (B) Schematic demonstrating the principle of FCCS. Simulated data and subsequent correlation curves for co-diffusing red and green particles (top) and for randomly diffusing particles (bottom) is shown. The amplitude of the cross-correlation relative to the auto-correlation curves is indicative of the strength of the interaction. (C) A line profile was generated spanning the NE and line-scanning FCCS data were collected in cells containing Ndc1-mTurquoise2 (green) and Mps3-YFP (red). Bar, 2 µm. (D) Each scan along the line can be visualized in a kymograph, which shows at each time point the fluctuations in molecules and complexes as they traverse the NE. Bar, 2 µm. Correlation analysis of intensity fluctuations was performed at the sites where the line-scan crossed the NE. (E) Average auto-correlation and cross-correlation curves and fits are shown for Ndc1-mTurquoise2 and Mps3-YFP in wild-type (SLJ7436) and pom152Δ (SLJ7438) cells. Error bars represent the SEM. Inset, magnification of plots to show differences in cross-correlation between strains. (F) The apparent fraction Mps3 and Ndc1 bound is increased in pom152Δ cells (P < 0.0001). Error bars represent Monte Carlo standard errors. (G and H) FCCS was also analyzed in wild-type (SLJ7835) and pom152Δ (SLJ7836) strains containing Ndc1-mTurquoise (green) and Nup49-YFP (red), and analyzed as in E and F. In H, the bound fraction presumably reflects the NPC.
Mentions: Using the MYTH system, we compared binding of Ndc1 and ndc1-L562S to Pom34, Mps3, and Nbp1 in the presence and absence of POM152 to better understand how Ndc1 is distributed between NE complexes. Deletion of POM152 did not affect binding of Pom34 or Nbp1 to either wild-type or the mutant versions of Ndc1 that we tested (Fig. 6 A). Pom152 was also not required for Ndc1 interaction with other proteins such as Yop1 and Nup59. (Fig. S3 B). However, pom152Δ significantly enhanced association of ndc1-L562S and Mps3 as well as increased binding of Ndc1 and Mps3 (Fig. 6 A), indicating that Pom152 and Mps3 likely act antagonistically to control the distribution of Ndc1.

Bottom Line: We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication.Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component.Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.

ABSTRACT
In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain-containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1-Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

Show MeSH
Related in: MedlinePlus