Limits...
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.

Show MeSH

Related in: MedlinePlus

The ndc1-L562S allele is lethal due to a defect in SPB duplication. (A) Growth of ndc1Δ (SLJ6064) cells containing NDC1 or the indicated alleles was tested by plating 10-fold serial dilutions of cells onto YPD or 5-FOA. Plates were incubated for 2 d at 30 and 37°C and for 3 d at 23°C. (B) Wild-type (SLJ001) cells or cells containing NDC1 or ndc1-L562S as well as GAL-NDC1-GFP (SLJ6367 or SLJ6369, respectively) were serially diluted onto YPGR or YPD and grown for 3 d at 30°C. (C–F) These same cells, or isogenic derivatives containing Spc42-mCherry and GFP-Tub1 (SLJ6847 or SLJ6847), were grown in YPGR at 30°C then were transferred into YPD to repress expression of NDC1-GFP. (C) Flow cytometric analysis of DNA content and budding index were used to assay ploidy and cell cycle arrest at the indicated times. Spindle morphology was examined using GFP-Tub1 (green) and Spc42-mCherry (red). Representative images from large-budded cells are shown in D, and the percentage of cells with bipolar and monopolar spindles was quantitated in E (n = 200). (F–I) Serial sections through nuclei of 19 NDC1 (SLJ6367) and 30 ndc1-L562S (SLJ6369) cells shifted YPD for 24 h were examined by EM: 15/19 and 3/30 nuclei from NDC1 and ndc1-L562S had bipolar spindles as depicted in F and G, respectively. In G, the second SPB found in an adjacent section is shown in the inset. A single SPB was found in 27/30 nuclei from ndc1-L562S, often on an NE invagination (H) or associated with material on the NE resembling an intermediate in SPB assembly (I, arrow). Nuclear pore complexes are marked with an asterisk. Bar, indicated. (J) NPC integrity was examined by Nup49-mCherry (red) in SLJ6822 and SLJ6823. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The ndc1-L562S allele is lethal due to a defect in SPB duplication. (A) Growth of ndc1Δ (SLJ6064) cells containing NDC1 or the indicated alleles was tested by plating 10-fold serial dilutions of cells onto YPD or 5-FOA. Plates were incubated for 2 d at 30 and 37°C and for 3 d at 23°C. (B) Wild-type (SLJ001) cells or cells containing NDC1 or ndc1-L562S as well as GAL-NDC1-GFP (SLJ6367 or SLJ6369, respectively) were serially diluted onto YPGR or YPD and grown for 3 d at 30°C. (C–F) These same cells, or isogenic derivatives containing Spc42-mCherry and GFP-Tub1 (SLJ6847 or SLJ6847), were grown in YPGR at 30°C then were transferred into YPD to repress expression of NDC1-GFP. (C) Flow cytometric analysis of DNA content and budding index were used to assay ploidy and cell cycle arrest at the indicated times. Spindle morphology was examined using GFP-Tub1 (green) and Spc42-mCherry (red). Representative images from large-budded cells are shown in D, and the percentage of cells with bipolar and monopolar spindles was quantitated in E (n = 200). (F–I) Serial sections through nuclei of 19 NDC1 (SLJ6367) and 30 ndc1-L562S (SLJ6369) cells shifted YPD for 24 h were examined by EM: 15/19 and 3/30 nuclei from NDC1 and ndc1-L562S had bipolar spindles as depicted in F and G, respectively. In G, the second SPB found in an adjacent section is shown in the inset. A single SPB was found in 27/30 nuclei from ndc1-L562S, often on an NE invagination (H) or associated with material on the NE resembling an intermediate in SPB assembly (I, arrow). Nuclear pore complexes are marked with an asterisk. Bar, indicated. (J) NPC integrity was examined by Nup49-mCherry (red) in SLJ6822 and SLJ6823. Bars, 2 µm.

Mentions: Mutation of 22 highly conserved residues did not affect Ndc1’s interaction with Pom152, Pom34, or Nbp1 based on the MYTH system (Fig. S2 A; Fig. S3 A). We anticipated that all of these alleles would be fully functional because they are able to associate with key Ndc1-binding proteins at both the SPB and NPC. However, two alleles, ndc1-L562S and ndc1-V340Q, were lethal at all temperatures (Fig. 3 A; Table 1). Further analysis of ndc1-L562S showed that it binds to two additional Ndc1-interacting partners at the NPC, Nup59 and Yop1 (Fig. S3B; Uetz et al., 2000; Casey et al., 2012).


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)

The ndc1-L562S allele is lethal due to a defect in SPB duplication. (A) Growth of ndc1Δ (SLJ6064) cells containing NDC1 or the indicated alleles was tested by plating 10-fold serial dilutions of cells onto YPD or 5-FOA. Plates were incubated for 2 d at 30 and 37°C and for 3 d at 23°C. (B) Wild-type (SLJ001) cells or cells containing NDC1 or ndc1-L562S as well as GAL-NDC1-GFP (SLJ6367 or SLJ6369, respectively) were serially diluted onto YPGR or YPD and grown for 3 d at 30°C. (C–F) These same cells, or isogenic derivatives containing Spc42-mCherry and GFP-Tub1 (SLJ6847 or SLJ6847), were grown in YPGR at 30°C then were transferred into YPD to repress expression of NDC1-GFP. (C) Flow cytometric analysis of DNA content and budding index were used to assay ploidy and cell cycle arrest at the indicated times. Spindle morphology was examined using GFP-Tub1 (green) and Spc42-mCherry (red). Representative images from large-budded cells are shown in D, and the percentage of cells with bipolar and monopolar spindles was quantitated in E (n = 200). (F–I) Serial sections through nuclei of 19 NDC1 (SLJ6367) and 30 ndc1-L562S (SLJ6369) cells shifted YPD for 24 h were examined by EM: 15/19 and 3/30 nuclei from NDC1 and ndc1-L562S had bipolar spindles as depicted in F and G, respectively. In G, the second SPB found in an adjacent section is shown in the inset. A single SPB was found in 27/30 nuclei from ndc1-L562S, often on an NE invagination (H) or associated with material on the NE resembling an intermediate in SPB assembly (I, arrow). Nuclear pore complexes are marked with an asterisk. Bar, indicated. (J) NPC integrity was examined by Nup49-mCherry (red) in SLJ6822 and SLJ6823. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The ndc1-L562S allele is lethal due to a defect in SPB duplication. (A) Growth of ndc1Δ (SLJ6064) cells containing NDC1 or the indicated alleles was tested by plating 10-fold serial dilutions of cells onto YPD or 5-FOA. Plates were incubated for 2 d at 30 and 37°C and for 3 d at 23°C. (B) Wild-type (SLJ001) cells or cells containing NDC1 or ndc1-L562S as well as GAL-NDC1-GFP (SLJ6367 or SLJ6369, respectively) were serially diluted onto YPGR or YPD and grown for 3 d at 30°C. (C–F) These same cells, or isogenic derivatives containing Spc42-mCherry and GFP-Tub1 (SLJ6847 or SLJ6847), were grown in YPGR at 30°C then were transferred into YPD to repress expression of NDC1-GFP. (C) Flow cytometric analysis of DNA content and budding index were used to assay ploidy and cell cycle arrest at the indicated times. Spindle morphology was examined using GFP-Tub1 (green) and Spc42-mCherry (red). Representative images from large-budded cells are shown in D, and the percentage of cells with bipolar and monopolar spindles was quantitated in E (n = 200). (F–I) Serial sections through nuclei of 19 NDC1 (SLJ6367) and 30 ndc1-L562S (SLJ6369) cells shifted YPD for 24 h were examined by EM: 15/19 and 3/30 nuclei from NDC1 and ndc1-L562S had bipolar spindles as depicted in F and G, respectively. In G, the second SPB found in an adjacent section is shown in the inset. A single SPB was found in 27/30 nuclei from ndc1-L562S, often on an NE invagination (H) or associated with material on the NE resembling an intermediate in SPB assembly (I, arrow). Nuclear pore complexes are marked with an asterisk. Bar, indicated. (J) NPC integrity was examined by Nup49-mCherry (red) in SLJ6822 and SLJ6823. Bars, 2 µm.
Mentions: Mutation of 22 highly conserved residues did not affect Ndc1’s interaction with Pom152, Pom34, or Nbp1 based on the MYTH system (Fig. S2 A; Fig. S3 A). We anticipated that all of these alleles would be fully functional because they are able to associate with key Ndc1-binding proteins at both the SPB and NPC. However, two alleles, ndc1-L562S and ndc1-V340Q, were lethal at all temperatures (Fig. 3 A; Table 1). Further analysis of ndc1-L562S showed that it binds to two additional Ndc1-interacting partners at the NPC, Nup59 and Yop1 (Fig. S3B; Uetz et al., 2000; Casey et al., 2012).

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