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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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Identification of new NDC1 alleles unable to bind to SPB and NPC components. (A) Schematic of MYTH system used to assay interaction with Ndc1. Prey proteins are fused to the N terminus of ubiquitin (NubG) and expressed along with the bait plasmid containing NDC1 fused to the C terminus of ubiquitin (Cub) and the LexA-VP16 transcription factor (TF) in yeast cells (SLJ5572) containing the reporter genes ADE2 and HIS3. If Ndc1 is able to interact with the prey, a functional ubiquitin is recognized by ubiquitin proteases that cleave the TF; the soluble TF activates gene expression, which is detected by cell growth on media lacking histidine and adenine. (B and C) Mid-log phase cells producing Ndc1-GFP from the endogenous locus (SLJ6881), the MYTH bait plasmid (SLJ7484), or from the GAL1 promoter (SLJ7848) were analyzed after a 2-h induction in 2% galactose/2% raffinose-containing media. In B, protein levels in whole-cell lysates were determined by Western blotting with anti-GFP antibodies. Pgk1 served as a loading control and allowed for normalization of the levels of the baits. The strain containing an empty vector (−) was assigned a value of 0 whereas the strain containing Ndc1-GFP expressed from the endogenous locus was given a value of 1. In C, the same strains were imaged to determine to localization of the Ndc1-GFP. Bar, 2 µm. (D and E) Prey plasmids containing the NPC components POM152 or POM34 or the SPB component NBP1 were tested in combination with bait plasmids containing no insert (vector), wild-type NDC1, or point mutations in NDC1 as indicated (see Fig. S1 and Fig. S2 A). ndc1-39 (T14M, F218V, L288M, E293G, M457T, and F643L) is a ts mutant defective in SPB duplication and NPC assembly (Lau et al., 2004). The presence of both bait and prey plasmids was detected on SD-Leu-Trp media, and activation of the reporters in MYTH was assayed on SD-Leu-Trp-His-Ade plus 3-AT, which reduces background by selecting for robust expression of HIS3. 10-fold serial dilutions of cells were spotted onto plates that were incubated for 3 d at 30°C and then placed at 4°C overnight. In E, mutants described in the text and summarized in Table 1 are highlighted. Blue is used for the allele that is unable to bind to Pom34, Pom152, Nbp1, and Mps3; red is used for alleles that are unable to bind Nbp1 and Mps3 but bind to Pom152 and Pom34. (F) Bait proteins were fused to GFP so that their expression and localization could be examined as in B.
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fig1: Identification of new NDC1 alleles unable to bind to SPB and NPC components. (A) Schematic of MYTH system used to assay interaction with Ndc1. Prey proteins are fused to the N terminus of ubiquitin (NubG) and expressed along with the bait plasmid containing NDC1 fused to the C terminus of ubiquitin (Cub) and the LexA-VP16 transcription factor (TF) in yeast cells (SLJ5572) containing the reporter genes ADE2 and HIS3. If Ndc1 is able to interact with the prey, a functional ubiquitin is recognized by ubiquitin proteases that cleave the TF; the soluble TF activates gene expression, which is detected by cell growth on media lacking histidine and adenine. (B and C) Mid-log phase cells producing Ndc1-GFP from the endogenous locus (SLJ6881), the MYTH bait plasmid (SLJ7484), or from the GAL1 promoter (SLJ7848) were analyzed after a 2-h induction in 2% galactose/2% raffinose-containing media. In B, protein levels in whole-cell lysates were determined by Western blotting with anti-GFP antibodies. Pgk1 served as a loading control and allowed for normalization of the levels of the baits. The strain containing an empty vector (−) was assigned a value of 0 whereas the strain containing Ndc1-GFP expressed from the endogenous locus was given a value of 1. In C, the same strains were imaged to determine to localization of the Ndc1-GFP. Bar, 2 µm. (D and E) Prey plasmids containing the NPC components POM152 or POM34 or the SPB component NBP1 were tested in combination with bait plasmids containing no insert (vector), wild-type NDC1, or point mutations in NDC1 as indicated (see Fig. S1 and Fig. S2 A). ndc1-39 (T14M, F218V, L288M, E293G, M457T, and F643L) is a ts mutant defective in SPB duplication and NPC assembly (Lau et al., 2004). The presence of both bait and prey plasmids was detected on SD-Leu-Trp media, and activation of the reporters in MYTH was assayed on SD-Leu-Trp-His-Ade plus 3-AT, which reduces background by selecting for robust expression of HIS3. 10-fold serial dilutions of cells were spotted onto plates that were incubated for 3 d at 30°C and then placed at 4°C overnight. In E, mutants described in the text and summarized in Table 1 are highlighted. Blue is used for the allele that is unable to bind to Pom34, Pom152, Nbp1, and Mps3; red is used for alleles that are unable to bind Nbp1 and Mps3 but bind to Pom152 and Pom34. (F) Bait proteins were fused to GFP so that their expression and localization could be examined as in B.

Mentions: To study the recruitment of Ndc1 to the SPB and NPC, we set up a membrane-based yeast two-hybrid (MYTH) system (Fig. 1 A; Stagljar and Fields, 2002; Thaminy et al., 2003; Snider et al., 2010). The bait, NDC1 (or mutant derivatives), was fused with the C terminus of ubiquitin (Cub) and expressed using the CYC1 promoter on a low-copy LEU2-marked centromeric plasmid. This resulted in low levels of Ndc1 expression at the NE compared with Ndc1 expressed from the chromosomal locus or a GAL1-driven version of NDC1-GFP (Fig. 1, B and C). The preys were fused with a mutant version of the N terminus of ubiquitin (NubG) that cannot associate with the C-terminal ubiquitin domain and expressed using the ADH1 promoter on a TRP1-marked plasmid.


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)

Identification of new NDC1 alleles unable to bind to SPB and NPC components. (A) Schematic of MYTH system used to assay interaction with Ndc1. Prey proteins are fused to the N terminus of ubiquitin (NubG) and expressed along with the bait plasmid containing NDC1 fused to the C terminus of ubiquitin (Cub) and the LexA-VP16 transcription factor (TF) in yeast cells (SLJ5572) containing the reporter genes ADE2 and HIS3. If Ndc1 is able to interact with the prey, a functional ubiquitin is recognized by ubiquitin proteases that cleave the TF; the soluble TF activates gene expression, which is detected by cell growth on media lacking histidine and adenine. (B and C) Mid-log phase cells producing Ndc1-GFP from the endogenous locus (SLJ6881), the MYTH bait plasmid (SLJ7484), or from the GAL1 promoter (SLJ7848) were analyzed after a 2-h induction in 2% galactose/2% raffinose-containing media. In B, protein levels in whole-cell lysates were determined by Western blotting with anti-GFP antibodies. Pgk1 served as a loading control and allowed for normalization of the levels of the baits. The strain containing an empty vector (−) was assigned a value of 0 whereas the strain containing Ndc1-GFP expressed from the endogenous locus was given a value of 1. In C, the same strains were imaged to determine to localization of the Ndc1-GFP. Bar, 2 µm. (D and E) Prey plasmids containing the NPC components POM152 or POM34 or the SPB component NBP1 were tested in combination with bait plasmids containing no insert (vector), wild-type NDC1, or point mutations in NDC1 as indicated (see Fig. S1 and Fig. S2 A). ndc1-39 (T14M, F218V, L288M, E293G, M457T, and F643L) is a ts mutant defective in SPB duplication and NPC assembly (Lau et al., 2004). The presence of both bait and prey plasmids was detected on SD-Leu-Trp media, and activation of the reporters in MYTH was assayed on SD-Leu-Trp-His-Ade plus 3-AT, which reduces background by selecting for robust expression of HIS3. 10-fold serial dilutions of cells were spotted onto plates that were incubated for 3 d at 30°C and then placed at 4°C overnight. In E, mutants described in the text and summarized in Table 1 are highlighted. Blue is used for the allele that is unable to bind to Pom34, Pom152, Nbp1, and Mps3; red is used for alleles that are unable to bind Nbp1 and Mps3 but bind to Pom152 and Pom34. (F) Bait proteins were fused to GFP so that their expression and localization could be examined as in B.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Identification of new NDC1 alleles unable to bind to SPB and NPC components. (A) Schematic of MYTH system used to assay interaction with Ndc1. Prey proteins are fused to the N terminus of ubiquitin (NubG) and expressed along with the bait plasmid containing NDC1 fused to the C terminus of ubiquitin (Cub) and the LexA-VP16 transcription factor (TF) in yeast cells (SLJ5572) containing the reporter genes ADE2 and HIS3. If Ndc1 is able to interact with the prey, a functional ubiquitin is recognized by ubiquitin proteases that cleave the TF; the soluble TF activates gene expression, which is detected by cell growth on media lacking histidine and adenine. (B and C) Mid-log phase cells producing Ndc1-GFP from the endogenous locus (SLJ6881), the MYTH bait plasmid (SLJ7484), or from the GAL1 promoter (SLJ7848) were analyzed after a 2-h induction in 2% galactose/2% raffinose-containing media. In B, protein levels in whole-cell lysates were determined by Western blotting with anti-GFP antibodies. Pgk1 served as a loading control and allowed for normalization of the levels of the baits. The strain containing an empty vector (−) was assigned a value of 0 whereas the strain containing Ndc1-GFP expressed from the endogenous locus was given a value of 1. In C, the same strains were imaged to determine to localization of the Ndc1-GFP. Bar, 2 µm. (D and E) Prey plasmids containing the NPC components POM152 or POM34 or the SPB component NBP1 were tested in combination with bait plasmids containing no insert (vector), wild-type NDC1, or point mutations in NDC1 as indicated (see Fig. S1 and Fig. S2 A). ndc1-39 (T14M, F218V, L288M, E293G, M457T, and F643L) is a ts mutant defective in SPB duplication and NPC assembly (Lau et al., 2004). The presence of both bait and prey plasmids was detected on SD-Leu-Trp media, and activation of the reporters in MYTH was assayed on SD-Leu-Trp-His-Ade plus 3-AT, which reduces background by selecting for robust expression of HIS3. 10-fold serial dilutions of cells were spotted onto plates that were incubated for 3 d at 30°C and then placed at 4°C overnight. In E, mutants described in the text and summarized in Table 1 are highlighted. Blue is used for the allele that is unable to bind to Pom34, Pom152, Nbp1, and Mps3; red is used for alleles that are unable to bind Nbp1 and Mps3 but bind to Pom152 and Pom34. (F) Bait proteins were fused to GFP so that their expression and localization could be examined as in B.
Mentions: To study the recruitment of Ndc1 to the SPB and NPC, we set up a membrane-based yeast two-hybrid (MYTH) system (Fig. 1 A; Stagljar and Fields, 2002; Thaminy et al., 2003; Snider et al., 2010). The bait, NDC1 (or mutant derivatives), was fused with the C terminus of ubiquitin (Cub) and expressed using the CYC1 promoter on a low-copy LEU2-marked centromeric plasmid. This resulted in low levels of Ndc1 expression at the NE compared with Ndc1 expressed from the chromosomal locus or a GAL1-driven version of NDC1-GFP (Fig. 1, B and C). The preys were fused with a mutant version of the N terminus of ubiquitin (NubG) that cannot associate with the C-terminal ubiquitin domain and expressed using the ADH1 promoter on a TRP1-marked plasmid.

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