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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 mutants exhibit the nibbled colony phenotype and ulp1NΔ338 is epistatic to mlp1Δ mlp2Δ for bleomycin sensitivity. (A) ulp1NΔ210 and ulp1NΔ338 strains grow as nibbled colonies on agar (top). This defect is cured by removal of 2-micron circle (bottom). (B) Mid-log phase YPD-grown cells were spotted at 10-fold serial dilutions (from 105 to 10) onto plates with or without bleomycin.
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fig4: ulp1 mutants exhibit the nibbled colony phenotype and ulp1NΔ338 is epistatic to mlp1Δ mlp2Δ for bleomycin sensitivity. (A) ulp1NΔ210 and ulp1NΔ338 strains grow as nibbled colonies on agar (top). This defect is cured by removal of 2-micron circle (bottom). (B) Mid-log phase YPD-grown cells were spotted at 10-fold serial dilutions (from 105 to 10) onto plates with or without bleomycin.

Mentions: To investigate the relationship between Ulp1 and 2-micron circle directly, we tested if delocalization of Ulp1 with its anchoring proteins intact can result in elevated levels of 2-micron circle and nibbled colonies. Ulp1 contains two separate domains: the COOH-terminal catalytic domain (residues 403–612) and the NH2-terminal regulatory domain (residues 1–340), which is necessary and sufficient for NPC localization (Li and Hochstrasser, 2003; Panse et al., 2003). It was previously shown that gradual deletions of the NH2-terminal domain lead to increased delocalization of Ulp1 from the nuclear rim without affecting its enzymatic activity (Li and Hochstrasser, 2003; Panse et al., 2003). Therefore, we examined the effect of two NH2-terminal deletion constructs, ulp1NΔ210 (residues 1–209 deleted) and ulp1NΔ338 (residues 1–337 deleted), on 2-micron circle and colony morphology. Ulp1NΔ210 contains some sequences required for nuclear rim localization, whereas ulp1NΔ338 lacks all sequences for docking (Panse et al., 2003). As shown in Fig. 4 A, ulp1NΔ210 and ulp1NΔ338 colonies are nibbled, and this defect is rescued by removal of 2-micron circle. Furthermore, the copy number of 2-micron circle in ulp1NΔ210 and ulp1NΔ338 mutants increases ∼2.5- and ∼4-fold, respectively (Fig. 1, B and C). Therefore, delocalization of Ulp1 by deleting its NH2 terminus results in higher levels of 2-micron circle, and the severity of delocalization is positively correlated with the levels of 2-micron circle.


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 mutants exhibit the nibbled colony phenotype and ulp1NΔ338 is epistatic to mlp1Δ mlp2Δ for bleomycin sensitivity. (A) ulp1NΔ210 and ulp1NΔ338 strains grow as nibbled colonies on agar (top). This defect is cured by removal of 2-micron circle (bottom). (B) Mid-log phase YPD-grown cells were spotted at 10-fold serial dilutions (from 105 to 10) onto plates with or without bleomycin.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: ulp1 mutants exhibit the nibbled colony phenotype and ulp1NΔ338 is epistatic to mlp1Δ mlp2Δ for bleomycin sensitivity. (A) ulp1NΔ210 and ulp1NΔ338 strains grow as nibbled colonies on agar (top). This defect is cured by removal of 2-micron circle (bottom). (B) Mid-log phase YPD-grown cells were spotted at 10-fold serial dilutions (from 105 to 10) onto plates with or without bleomycin.
Mentions: To investigate the relationship between Ulp1 and 2-micron circle directly, we tested if delocalization of Ulp1 with its anchoring proteins intact can result in elevated levels of 2-micron circle and nibbled colonies. Ulp1 contains two separate domains: the COOH-terminal catalytic domain (residues 403–612) and the NH2-terminal regulatory domain (residues 1–340), which is necessary and sufficient for NPC localization (Li and Hochstrasser, 2003; Panse et al., 2003). It was previously shown that gradual deletions of the NH2-terminal domain lead to increased delocalization of Ulp1 from the nuclear rim without affecting its enzymatic activity (Li and Hochstrasser, 2003; Panse et al., 2003). Therefore, we examined the effect of two NH2-terminal deletion constructs, ulp1NΔ210 (residues 1–209 deleted) and ulp1NΔ338 (residues 1–337 deleted), on 2-micron circle and colony morphology. Ulp1NΔ210 contains some sequences required for nuclear rim localization, whereas ulp1NΔ338 lacks all sequences for docking (Panse et al., 2003). As shown in Fig. 4 A, ulp1NΔ210 and ulp1NΔ338 colonies are nibbled, and this defect is rescued by removal of 2-micron circle. Furthermore, the copy number of 2-micron circle in ulp1NΔ210 and ulp1NΔ338 mutants increases ∼2.5- and ∼4-fold, respectively (Fig. 1, B and C). Therefore, delocalization of Ulp1 by deleting its NH2 terminus results in higher levels of 2-micron circle, and the severity of delocalization is positively correlated with the levels of 2-micron circle.

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