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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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Ulp1 levels and the SUMO conjugate pattern are changed in cells lacking Mlps or Nup60. (A) Total protein was extracted from indicated strains and subjected to immunoblot analysis using anti-GFP antibody. ULP1-YFP was used to replace the endogenous ULP1 in all strains, except in WT (untagged). The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. In the bottom panel, Ulp1-YFP and PGK bands were quantified and the relative amount of Ulp1 was calculated as the amount of Ulp1-YFP signals divided by PGK signals. The value for wild-type strains was considered to be 1. Error bars indicate SDs from three similar blots. (B) Immunoblot analysis of yeast lysates prepared from indicated strains was performed using anti-SUMO antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. Arrows indicate the SUMO conjugates whose levels increase in mlp1Δ mlp2Δ and nup60Δ strains. The asterisks mark the SUMO conjugates whose levels diminish in mlp1Δ, mlp2Δ, mlp1Δ mlp2Δ, and nup60Δ strains.
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fig3: Ulp1 levels and the SUMO conjugate pattern are changed in cells lacking Mlps or Nup60. (A) Total protein was extracted from indicated strains and subjected to immunoblot analysis using anti-GFP antibody. ULP1-YFP was used to replace the endogenous ULP1 in all strains, except in WT (untagged). The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. In the bottom panel, Ulp1-YFP and PGK bands were quantified and the relative amount of Ulp1 was calculated as the amount of Ulp1-YFP signals divided by PGK signals. The value for wild-type strains was considered to be 1. Error bars indicate SDs from three similar blots. (B) Immunoblot analysis of yeast lysates prepared from indicated strains was performed using anti-SUMO antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. Arrows indicate the SUMO conjugates whose levels increase in mlp1Δ mlp2Δ and nup60Δ strains. The asterisks mark the SUMO conjugates whose levels diminish in mlp1Δ, mlp2Δ, mlp1Δ mlp2Δ, and nup60Δ strains.

Mentions: To explore further the relationship between Mlps and Ulp1, we examined the localization of Ulp1-YFP in mlp1Δ, mlp2Δ, and mlp1Δ mlp2Δ cells. We observed that the signal intensity of Ulp1-YFP at the nuclear rim decreases moderately in mlp1Δ or mlp2Δ strains compared with wild-type strains (Fig. 2 B). Because the protein level of Ulp1-YFP in these mutants is the same as in wild-type strains (Fig. 3 A), the reduction of nuclear rim signals suggests that some Ulp1 proteins are delocalized. In mlp1Δ mlp2Δ double mutants, Ulp1-YFP signals at the nuclear rim are greatly reduced (Fig. 2 B). In addition, this is associated with a fivefold reduction of Ulp1 protein levels (Fig. 3 A). These results indicate that lacking both Mlp1 and Mlp2 synergistically affects Ulp1 protein levels and localization. The simplest explanation is that severe delocalization of Ulp1 due to the lack of both Mlps leads to the reduced protein levels.


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)

Ulp1 levels and the SUMO conjugate pattern are changed in cells lacking Mlps or Nup60. (A) Total protein was extracted from indicated strains and subjected to immunoblot analysis using anti-GFP antibody. ULP1-YFP was used to replace the endogenous ULP1 in all strains, except in WT (untagged). The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. In the bottom panel, Ulp1-YFP and PGK bands were quantified and the relative amount of Ulp1 was calculated as the amount of Ulp1-YFP signals divided by PGK signals. The value for wild-type strains was considered to be 1. Error bars indicate SDs from three similar blots. (B) Immunoblot analysis of yeast lysates prepared from indicated strains was performed using anti-SUMO antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. Arrows indicate the SUMO conjugates whose levels increase in mlp1Δ mlp2Δ and nup60Δ strains. The asterisks mark the SUMO conjugates whose levels diminish in mlp1Δ, mlp2Δ, mlp1Δ mlp2Δ, and nup60Δ strains.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Ulp1 levels and the SUMO conjugate pattern are changed in cells lacking Mlps or Nup60. (A) Total protein was extracted from indicated strains and subjected to immunoblot analysis using anti-GFP antibody. ULP1-YFP was used to replace the endogenous ULP1 in all strains, except in WT (untagged). The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. In the bottom panel, Ulp1-YFP and PGK bands were quantified and the relative amount of Ulp1 was calculated as the amount of Ulp1-YFP signals divided by PGK signals. The value for wild-type strains was considered to be 1. Error bars indicate SDs from three similar blots. (B) Immunoblot analysis of yeast lysates prepared from indicated strains was performed using anti-SUMO antibody. The same blot was stripped and reprobed with anti-PGK antibody to check loading consistency. Arrows indicate the SUMO conjugates whose levels increase in mlp1Δ mlp2Δ and nup60Δ strains. The asterisks mark the SUMO conjugates whose levels diminish in mlp1Δ, mlp2Δ, mlp1Δ mlp2Δ, and nup60Δ strains.
Mentions: To explore further the relationship between Mlps and Ulp1, we examined the localization of Ulp1-YFP in mlp1Δ, mlp2Δ, and mlp1Δ mlp2Δ cells. We observed that the signal intensity of Ulp1-YFP at the nuclear rim decreases moderately in mlp1Δ or mlp2Δ strains compared with wild-type strains (Fig. 2 B). Because the protein level of Ulp1-YFP in these mutants is the same as in wild-type strains (Fig. 3 A), the reduction of nuclear rim signals suggests that some Ulp1 proteins are delocalized. In mlp1Δ mlp2Δ double mutants, Ulp1-YFP signals at the nuclear rim are greatly reduced (Fig. 2 B). In addition, this is associated with a fivefold reduction of Ulp1 protein levels (Fig. 3 A). These results indicate that lacking both Mlp1 and Mlp2 synergistically affects Ulp1 protein levels and localization. The simplest explanation is that severe delocalization of Ulp1 due to the lack of both Mlps leads to the reduced protein levels.

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