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Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality.

Zhao X, Wu CY, Blobel G - J. Cell Biol. (2004)

Bottom Line: Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1.Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope.Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

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Overexpression of Ulp1 restores its localization at the nuclear envelope and rescues the nibbled colony morphology as well as the bleomycin sensitivity of mlp1Δ mlp2Δ strains. (A) Ulp1 proteins, tagged with GFP, were expressed from the ULP1 promoter (Ulp1) or from the NOP1 promoter on a CEN plasmid (pULP1). In the latter case, the endogenous ULP1 gene was deleted. Total protein was extracted and subjected to immunoblot analysis using polyclonal anti-GFP antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. (B) Ulp1 tagged with GFP was expressed from the NOP1 promoter in ulp1Δ or mlp1Δ mlp2Δ ulp1Δ strains. Fluorescent images of Ulp1-GFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. Bar, 2 μm. (C) The mlp1Δ mlp2Δ strain was transformed with pULP1 or with a vector. Transformants were streaked out on selective medium and grown at 30°C for 3 d before being photographed. (D) Sensitivity to bleomycin (5 μg/ml) was tested for the mlp1Δ mlp2Δ ulp1Δ strain containing pULP1, the mlp1Δ mlp2Δ strain containing the vector, and the wild-type strain containing the vector. Mid-log phase SC-Leu–grown cells were spotted at 10-fold serial dilutions (from 105 to 10 cells) onto plates with or without bleomycin.
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fig5: Overexpression of Ulp1 restores its localization at the nuclear envelope and rescues the nibbled colony morphology as well as the bleomycin sensitivity of mlp1Δ mlp2Δ strains. (A) Ulp1 proteins, tagged with GFP, were expressed from the ULP1 promoter (Ulp1) or from the NOP1 promoter on a CEN plasmid (pULP1). In the latter case, the endogenous ULP1 gene was deleted. Total protein was extracted and subjected to immunoblot analysis using polyclonal anti-GFP antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. (B) Ulp1 tagged with GFP was expressed from the NOP1 promoter in ulp1Δ or mlp1Δ mlp2Δ ulp1Δ strains. Fluorescent images of Ulp1-GFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. Bar, 2 μm. (C) The mlp1Δ mlp2Δ strain was transformed with pULP1 or with a vector. Transformants were streaked out on selective medium and grown at 30°C for 3 d before being photographed. (D) Sensitivity to bleomycin (5 μg/ml) was tested for the mlp1Δ mlp2Δ ulp1Δ strain containing pULP1, the mlp1Δ mlp2Δ strain containing the vector, and the wild-type strain containing the vector. Mid-log phase SC-Leu–grown cells were spotted at 10-fold serial dilutions (from 105 to 10 cells) onto plates with or without bleomycin.

Mentions: It is noteworthy that other tethering sites for Ulp1 exist, as residual Ulp1-YFP signals were seen at the nuclear rim in cells lacking Mlps (Fig. 2 B). The fact that these sites cannot substitute for Mlps to dock Ulp1 when Ulp1 is expressed at the endogenous level suggests that they might be less efficient. Should this be the case, they might be able to dock a sufficient amount of Ulp1 when Ulp1 is overexpressed. We found that Ulp1 exhibits continuous nuclear envelope localization, including the region juxtaposed to the nucleolus when it is moderately overexpressed from the NOP1 promoter, (Fig. 5, A and B). This is different from the Mlp-dependent localization pattern, which is punctuate and excluded from the nucleolus, indicating that alternative tethering of Ulp1 occurs. Consistently, this localization pattern is independent of Mlps (Fig. 5 B). The restoration of Ulp1 localization under overexpression conditions can rescue the nibbled colony morphology and bleomycin sensitivity of mlp1Δ mlp2Δ strains (Fig. 5, C and D). This result confirms the conclusion from our epistatic analysis and strongly supports the notion that the nibbled colony morphology and bleomycin sensitivity in mlp1Δ mlp2Δ strains are due to defects in regulating Ulp1.


Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality.

Zhao X, Wu CY, Blobel G - J. Cell Biol. (2004)

Overexpression of Ulp1 restores its localization at the nuclear envelope and rescues the nibbled colony morphology as well as the bleomycin sensitivity of mlp1Δ mlp2Δ strains. (A) Ulp1 proteins, tagged with GFP, were expressed from the ULP1 promoter (Ulp1) or from the NOP1 promoter on a CEN plasmid (pULP1). In the latter case, the endogenous ULP1 gene was deleted. Total protein was extracted and subjected to immunoblot analysis using polyclonal anti-GFP antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. (B) Ulp1 tagged with GFP was expressed from the NOP1 promoter in ulp1Δ or mlp1Δ mlp2Δ ulp1Δ strains. Fluorescent images of Ulp1-GFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. Bar, 2 μm. (C) The mlp1Δ mlp2Δ strain was transformed with pULP1 or with a vector. Transformants were streaked out on selective medium and grown at 30°C for 3 d before being photographed. (D) Sensitivity to bleomycin (5 μg/ml) was tested for the mlp1Δ mlp2Δ ulp1Δ strain containing pULP1, the mlp1Δ mlp2Δ strain containing the vector, and the wild-type strain containing the vector. Mid-log phase SC-Leu–grown cells were spotted at 10-fold serial dilutions (from 105 to 10 cells) onto plates with or without bleomycin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172573&req=5

fig5: Overexpression of Ulp1 restores its localization at the nuclear envelope and rescues the nibbled colony morphology as well as the bleomycin sensitivity of mlp1Δ mlp2Δ strains. (A) Ulp1 proteins, tagged with GFP, were expressed from the ULP1 promoter (Ulp1) or from the NOP1 promoter on a CEN plasmid (pULP1). In the latter case, the endogenous ULP1 gene was deleted. Total protein was extracted and subjected to immunoblot analysis using polyclonal anti-GFP antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. (B) Ulp1 tagged with GFP was expressed from the NOP1 promoter in ulp1Δ or mlp1Δ mlp2Δ ulp1Δ strains. Fluorescent images of Ulp1-GFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. Bar, 2 μm. (C) The mlp1Δ mlp2Δ strain was transformed with pULP1 or with a vector. Transformants were streaked out on selective medium and grown at 30°C for 3 d before being photographed. (D) Sensitivity to bleomycin (5 μg/ml) was tested for the mlp1Δ mlp2Δ ulp1Δ strain containing pULP1, the mlp1Δ mlp2Δ strain containing the vector, and the wild-type strain containing the vector. Mid-log phase SC-Leu–grown cells were spotted at 10-fold serial dilutions (from 105 to 10 cells) onto plates with or without bleomycin.
Mentions: It is noteworthy that other tethering sites for Ulp1 exist, as residual Ulp1-YFP signals were seen at the nuclear rim in cells lacking Mlps (Fig. 2 B). The fact that these sites cannot substitute for Mlps to dock Ulp1 when Ulp1 is expressed at the endogenous level suggests that they might be less efficient. Should this be the case, they might be able to dock a sufficient amount of Ulp1 when Ulp1 is overexpressed. We found that Ulp1 exhibits continuous nuclear envelope localization, including the region juxtaposed to the nucleolus when it is moderately overexpressed from the NOP1 promoter, (Fig. 5, A and B). This is different from the Mlp-dependent localization pattern, which is punctuate and excluded from the nucleolus, indicating that alternative tethering of Ulp1 occurs. Consistently, this localization pattern is independent of Mlps (Fig. 5 B). The restoration of Ulp1 localization under overexpression conditions can rescue the nibbled colony morphology and bleomycin sensitivity of mlp1Δ mlp2Δ strains (Fig. 5, C and D). This result confirms the conclusion from our epistatic analysis and strongly supports the notion that the nibbled colony morphology and bleomycin sensitivity in mlp1Δ mlp2Δ strains are due to defects in regulating Ulp1.

Bottom Line: Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1.Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope.Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

Show MeSH
Related in: MedlinePlus