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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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mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (A) 2-micron circle causes the nibbled colony morphology in mlp1Δ mlp2Δ strains. An mlp1Δ mlp2Δ strain exhibits the nibbled colony morphology (left). This defect is cured by removal of 2-micron circle (middle). Colonies become nibbled again after reintroduction of 2-micron circle to the mlp1Δ mlp2Δ cir0 strain (right). (B and C) mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (B) Genomic blot of total DNA extracted from indicated strains was hybridized with a probe specific to the 2-micron circle DNA sequence. rDNA was used as a loading control. (C) 2-micron circle DNA and rDNA bands in B and two additional blots were quantified. The relative amount of 2-micron circle DNA was calculated as the amount of 2-micron circle DNA signal divided by that of rDNA. The relative amount of 2-micron circle DNA in wild-type strains was considered to be 1.
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fig1: mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (A) 2-micron circle causes the nibbled colony morphology in mlp1Δ mlp2Δ strains. An mlp1Δ mlp2Δ strain exhibits the nibbled colony morphology (left). This defect is cured by removal of 2-micron circle (middle). Colonies become nibbled again after reintroduction of 2-micron circle to the mlp1Δ mlp2Δ cir0 strain (right). (B and C) mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (B) Genomic blot of total DNA extracted from indicated strains was hybridized with a probe specific to the 2-micron circle DNA sequence. rDNA was used as a loading control. (C) 2-micron circle DNA and rDNA bands in B and two additional blots were quantified. The relative amount of 2-micron circle DNA was calculated as the amount of 2-micron circle DNA signal divided by that of rDNA. The relative amount of 2-micron circle DNA in wild-type strains was considered to be 1.

Mentions: Our initial genetic analysis revealed that the nibbled phenotype of mlp1Δ mlp2Δ is not stably inherited, suggesting that it is likely caused by extrachromosomal or epigenetic factors (see Online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200405168/DC1). It was reported that high levels of 2-micron circle, an extrachromosomal plasmid found in most strains of Saccharomyces cerevisiae, can result in nibbled colonies (Broach and Volkert, 1991). 2-micron circle is packed into nucleosomes, and replicates and segregates using the same proteins as chromosomal DNA (Broach and Volkert, 1991; Mehta et al., 2002). It normally neither benefits nor harms the host when maintained at 50–100 copies per cell. Nevertheless, overproduction of 2-micron circle by overexpressing proteins unique to the plasmid's amplification system causes clonal lethality and nibbled colonies (Reynolds et al., 1987; Rose and Broach, 1990). A plausible explanation is that increased levels of 2-micron circle can titrate out essential replication and segregation machineries because even a twofold increase can give rise to DNA equivalent to two small yeast chromosomes. To test whether the nibbled phenotype of mlp1Δ mlp2Δ strains is related to 2-micron circle, we first removed the plasmid and observed that mlp1Δ mlp2Δ colonies are completely smooth and uniform in size (Fig. 1 A). In fact, the growth of mlp1Δ mlp2Δ cells that lack 2-micron circle (cir0) is indistinguishable from that of wild-type cells. Next, we reintroduced 2-micron circle to mlp1Δ mlp2Δ cir0 strains. The resulting mlp1Δ mlp2Δ strains containing the plasmid (cir+) regained the nibbled colony morphology (Fig. 1 A). Finally, we found that the copy number of 2-micron circle in mlp1Δ mlp2Δ, but not in either single mutants, is ∼2.5-fold higher than in the wild type (Fig. 1, B and C). These results reveal that an elevated level of 2-micron circle is the cause for the nibbled colony morphology of mlp1Δ mlp2Δ.


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)

mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (A) 2-micron circle causes the nibbled colony morphology in mlp1Δ mlp2Δ strains. An mlp1Δ mlp2Δ strain exhibits the nibbled colony morphology (left). This defect is cured by removal of 2-micron circle (middle). Colonies become nibbled again after reintroduction of 2-micron circle to the mlp1Δ mlp2Δ cir0 strain (right). (B and C) mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (B) Genomic blot of total DNA extracted from indicated strains was hybridized with a probe specific to the 2-micron circle DNA sequence. rDNA was used as a loading control. (C) 2-micron circle DNA and rDNA bands in B and two additional blots were quantified. The relative amount of 2-micron circle DNA was calculated as the amount of 2-micron circle DNA signal divided by that of rDNA. The relative amount of 2-micron circle DNA in wild-type strains was considered to be 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172573&req=5

fig1: mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (A) 2-micron circle causes the nibbled colony morphology in mlp1Δ mlp2Δ strains. An mlp1Δ mlp2Δ strain exhibits the nibbled colony morphology (left). This defect is cured by removal of 2-micron circle (middle). Colonies become nibbled again after reintroduction of 2-micron circle to the mlp1Δ mlp2Δ cir0 strain (right). (B and C) mlp1Δ mlp2Δ and ulp1 mutants contain elevated levels of 2-micron circle. (B) Genomic blot of total DNA extracted from indicated strains was hybridized with a probe specific to the 2-micron circle DNA sequence. rDNA was used as a loading control. (C) 2-micron circle DNA and rDNA bands in B and two additional blots were quantified. The relative amount of 2-micron circle DNA was calculated as the amount of 2-micron circle DNA signal divided by that of rDNA. The relative amount of 2-micron circle DNA in wild-type strains was considered to be 1.
Mentions: Our initial genetic analysis revealed that the nibbled phenotype of mlp1Δ mlp2Δ is not stably inherited, suggesting that it is likely caused by extrachromosomal or epigenetic factors (see Online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200405168/DC1). It was reported that high levels of 2-micron circle, an extrachromosomal plasmid found in most strains of Saccharomyces cerevisiae, can result in nibbled colonies (Broach and Volkert, 1991). 2-micron circle is packed into nucleosomes, and replicates and segregates using the same proteins as chromosomal DNA (Broach and Volkert, 1991; Mehta et al., 2002). It normally neither benefits nor harms the host when maintained at 50–100 copies per cell. Nevertheless, overproduction of 2-micron circle by overexpressing proteins unique to the plasmid's amplification system causes clonal lethality and nibbled colonies (Reynolds et al., 1987; Rose and Broach, 1990). A plausible explanation is that increased levels of 2-micron circle can titrate out essential replication and segregation machineries because even a twofold increase can give rise to DNA equivalent to two small yeast chromosomes. To test whether the nibbled phenotype of mlp1Δ mlp2Δ strains is related to 2-micron circle, we first removed the plasmid and observed that mlp1Δ mlp2Δ colonies are completely smooth and uniform in size (Fig. 1 A). In fact, the growth of mlp1Δ mlp2Δ cells that lack 2-micron circle (cir0) is indistinguishable from that of wild-type cells. Next, we reintroduced 2-micron circle to mlp1Δ mlp2Δ cir0 strains. The resulting mlp1Δ mlp2Δ strains containing the plasmid (cir+) regained the nibbled colony morphology (Fig. 1 A). Finally, we found that the copy number of 2-micron circle in mlp1Δ mlp2Δ, but not in either single mutants, is ∼2.5-fold higher than in the wild type (Fig. 1, B and C). These results reveal that an elevated level of 2-micron circle is the cause for the nibbled colony morphology of mlp1Δ mlp2Δ.

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