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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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Mlps and Nup60, but not Nup42, Nup53, or Nup85, are required to dock Ulp1 at the nuclear periphery. (A) The distribution of Mlps and Ulp1 is different from that of Nic96. Mlp1, Mlp2, and Ulp1 were tagged with YFP, whereas Nic96 was tagged with CFP. A nucleolar protein Nop1 was tagged with mRFP. Representative live-cell images of CFP, YFP, and mRFP fusion proteins as well as their merged pictures are shown. In all panels, mRFP fusion proteins are pseudocolored as red, YFP fusion proteins as green, and CFP fusion proteins as blue. Signals of Mlp1, Mlp2, and Ulp1, but not Nic96, are absent from the region of the nuclear rim juxtaposed to the nucleolus. (B) Mlps and Nup60 are required to dock Ulp1 at NPCs. Fluorescent images of Ulp1-YFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. All the images were acquired using the same settings for the camera and the microscope. Bars, 2 μm.
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fig2: Mlps and Nup60, but not Nup42, Nup53, or Nup85, are required to dock Ulp1 at the nuclear periphery. (A) The distribution of Mlps and Ulp1 is different from that of Nic96. Mlp1, Mlp2, and Ulp1 were tagged with YFP, whereas Nic96 was tagged with CFP. A nucleolar protein Nop1 was tagged with mRFP. Representative live-cell images of CFP, YFP, and mRFP fusion proteins as well as their merged pictures are shown. In all panels, mRFP fusion proteins are pseudocolored as red, YFP fusion proteins as green, and CFP fusion proteins as blue. Signals of Mlp1, Mlp2, and Ulp1, but not Nic96, are absent from the region of the nuclear rim juxtaposed to the nucleolus. (B) Mlps and Nup60 are required to dock Ulp1 at NPCs. Fluorescent images of Ulp1-YFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. All the images were acquired using the same settings for the camera and the microscope. Bars, 2 μm.

Mentions: To understand how Mlps affect 2-micron circle levels, we looked for other mutants that also exhibit nibbled colony morphology. As mentioned earlier, strains defective in a desumoylating enzyme, Ulp1, display a similar phenotype. The similar localization pattern of Mlps and Ulp1, as well as the common defects in colony morphology of their mutants, suggest that they may function in the same pathway. One possibility is that Mlps are required to dock Ulp1 at NPCs. Fluorescence microscopic examination of live cells showed that the localization pattern of Mlps differs from that of nucleoporins. Consistent with a recent report (Galy et al., 2004), nucleoporins are distributed all around the nuclear envelope, whereas Mlps are excluded from the region juxtaposed to the nucleolus (Fig. 2 A). We reasoned that if Mlps provide docking sites for Ulp1, Ulp1 should exhibit the same asymmetric distribution. To test this idea, Ulp1-YFP fusion protein was expressed in cells containing CFP-tagged nucleoporin Nic96 and mRFP-tagged nucleolar protein Nop1. Examination by fluorescence microscopy revealed that Ulp1-YFP is also excluded from the nucleolus-proximal region of the nuclear envelope (Fig. 2 A). Therefore, the localization pattern of Ulp1 resembles that of Mlps.


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)

Mlps and Nup60, but not Nup42, Nup53, or Nup85, are required to dock Ulp1 at the nuclear periphery. (A) The distribution of Mlps and Ulp1 is different from that of Nic96. Mlp1, Mlp2, and Ulp1 were tagged with YFP, whereas Nic96 was tagged with CFP. A nucleolar protein Nop1 was tagged with mRFP. Representative live-cell images of CFP, YFP, and mRFP fusion proteins as well as their merged pictures are shown. In all panels, mRFP fusion proteins are pseudocolored as red, YFP fusion proteins as green, and CFP fusion proteins as blue. Signals of Mlp1, Mlp2, and Ulp1, but not Nic96, are absent from the region of the nuclear rim juxtaposed to the nucleolus. (B) Mlps and Nup60 are required to dock Ulp1 at NPCs. Fluorescent images of Ulp1-YFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. All the images were acquired using the same settings for the camera and the microscope. Bars, 2 μm.
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Related In: Results  -  Collection

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fig2: Mlps and Nup60, but not Nup42, Nup53, or Nup85, are required to dock Ulp1 at the nuclear periphery. (A) The distribution of Mlps and Ulp1 is different from that of Nic96. Mlp1, Mlp2, and Ulp1 were tagged with YFP, whereas Nic96 was tagged with CFP. A nucleolar protein Nop1 was tagged with mRFP. Representative live-cell images of CFP, YFP, and mRFP fusion proteins as well as their merged pictures are shown. In all panels, mRFP fusion proteins are pseudocolored as red, YFP fusion proteins as green, and CFP fusion proteins as blue. Signals of Mlp1, Mlp2, and Ulp1, but not Nic96, are absent from the region of the nuclear rim juxtaposed to the nucleolus. (B) Mlps and Nup60 are required to dock Ulp1 at NPCs. Fluorescent images of Ulp1-YFP in live cells were taken at 14 Z-sections (step size = 0.3 μm), and the center sections are shown. All the images were acquired using the same settings for the camera and the microscope. Bars, 2 μm.
Mentions: To understand how Mlps affect 2-micron circle levels, we looked for other mutants that also exhibit nibbled colony morphology. As mentioned earlier, strains defective in a desumoylating enzyme, Ulp1, display a similar phenotype. The similar localization pattern of Mlps and Ulp1, as well as the common defects in colony morphology of their mutants, suggest that they may function in the same pathway. One possibility is that Mlps are required to dock Ulp1 at NPCs. Fluorescence microscopic examination of live cells showed that the localization pattern of Mlps differs from that of nucleoporins. Consistent with a recent report (Galy et al., 2004), nucleoporins are distributed all around the nuclear envelope, whereas Mlps are excluded from the region juxtaposed to the nucleolus (Fig. 2 A). We reasoned that if Mlps provide docking sites for Ulp1, Ulp1 should exhibit the same asymmetric distribution. To test this idea, Ulp1-YFP fusion protein was expressed in cells containing CFP-tagged nucleoporin Nic96 and mRFP-tagged nucleolar protein Nop1. Examination by fluorescence microscopy revealed that Ulp1-YFP is also excluded from the nucleolus-proximal region of the nuclear envelope (Fig. 2 A). Therefore, the localization pattern of Ulp1 resembles that of Mlps.

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