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Cooperation of sumoylated chromosomal proteins in rDNA maintenance.

Takahashi Y, Dulev S, Liu X, Hiller NJ, Zhao X, Strunnikov A - PLoS Genet. (2008)

Bottom Line: Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants.Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p.In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA.

ABSTRACT
SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

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Smt3p conjugates are enriched in the nucleolus.(A) Alternative tagging of Smt3p expressed at the native levels enables the identification of sumoylated proteins. Total sumoylated proteins were purified by IMAC from the strains expressing wild-type levels of poly-His/FLAG-tagged Smt3p (HF-Smt3, 924-YPH499b), poly-His/FLAG/S-tag-Smt3p (HFS-Smt3, 1008-YPH499), and poly-His/FLAG/GFP-Smt3p (HFG-Smt3, 1014-YPH499. Sumoylated proteins here and thereafter are separated by PAGE and detected by Western-blotting using the anti-FLAG antibody. Arrows indicate the proportional size shifts between the free SUMO forms. Molecular weight markers (×1000) are shown on the left. (B) GFP-SUMO localization as a function of conjugation/de-conjugation. The wild type (1014-YPH499b), ubc9-1 (1cYT630), and slx5Δ (1dYT631) strains expressing GFP-Smt3p as in (A) were incubated at 32°C (semi-permissive for ubc9-1) for 5 h and imaged live. The insert shows gradient-like distribution of SUMO typical for wild type, which is noticeable at higher magnifications. Scale bars here and elsewhere are 5 µm. (C) SUMO conjugates are concentrated in the nucleolus in slx8Δ cells. The wild type (1014-YPH499) and slx8Δ (1aYT629) strains co-expressing GFP-Smt3p and Nop1p-mRFP were incubated at 30°C; cell images were captured live.
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pgen-1000215-g001: Smt3p conjugates are enriched in the nucleolus.(A) Alternative tagging of Smt3p expressed at the native levels enables the identification of sumoylated proteins. Total sumoylated proteins were purified by IMAC from the strains expressing wild-type levels of poly-His/FLAG-tagged Smt3p (HF-Smt3, 924-YPH499b), poly-His/FLAG/S-tag-Smt3p (HFS-Smt3, 1008-YPH499), and poly-His/FLAG/GFP-Smt3p (HFG-Smt3, 1014-YPH499. Sumoylated proteins here and thereafter are separated by PAGE and detected by Western-blotting using the anti-FLAG antibody. Arrows indicate the proportional size shifts between the free SUMO forms. Molecular weight markers (×1000) are shown on the left. (B) GFP-SUMO localization as a function of conjugation/de-conjugation. The wild type (1014-YPH499b), ubc9-1 (1cYT630), and slx5Δ (1dYT631) strains expressing GFP-Smt3p as in (A) were incubated at 32°C (semi-permissive for ubc9-1) for 5 h and imaged live. The insert shows gradient-like distribution of SUMO typical for wild type, which is noticeable at higher magnifications. Scale bars here and elsewhere are 5 µm. (C) SUMO conjugates are concentrated in the nucleolus in slx8Δ cells. The wild type (1014-YPH499) and slx8Δ (1aYT629) strains co-expressing GFP-Smt3p and Nop1p-mRFP were incubated at 30°C; cell images were captured live.

Mentions: We recently showed that the bulk of sumoylated proteins are concentrated in a subnucleolar area reminiscent of rDNA chromatin, if the nuclear desumoylation enzyme Smt4p is inactivated by the smt4 gene deletion [11]. This observation suggested that the SUMO pathway may play a major role in the nucleolus. Therefore, we closely examined strains expressing GFP-Smt3p under the native SMT3 promoter, as a sole source of SUMO. To monitor the GFP-Smt3p (HFG-Smt3) modification biochemically we also generated a shorter fusion (with the extended S-tag, HFS-Smt3). Similar patterns of conjugated protein bands were observed for both strains, except bands shifted accordingly to the tag size (Figure 1A). Both the HFS-Smt3 and HFG-Smt3 strains had the wild type doubling time (not shown), indicating that HFS-Smt3p and HFG-Smt3p fulfill key functions of SUMO.


Cooperation of sumoylated chromosomal proteins in rDNA maintenance.

Takahashi Y, Dulev S, Liu X, Hiller NJ, Zhao X, Strunnikov A - PLoS Genet. (2008)

Smt3p conjugates are enriched in the nucleolus.(A) Alternative tagging of Smt3p expressed at the native levels enables the identification of sumoylated proteins. Total sumoylated proteins were purified by IMAC from the strains expressing wild-type levels of poly-His/FLAG-tagged Smt3p (HF-Smt3, 924-YPH499b), poly-His/FLAG/S-tag-Smt3p (HFS-Smt3, 1008-YPH499), and poly-His/FLAG/GFP-Smt3p (HFG-Smt3, 1014-YPH499. Sumoylated proteins here and thereafter are separated by PAGE and detected by Western-blotting using the anti-FLAG antibody. Arrows indicate the proportional size shifts between the free SUMO forms. Molecular weight markers (×1000) are shown on the left. (B) GFP-SUMO localization as a function of conjugation/de-conjugation. The wild type (1014-YPH499b), ubc9-1 (1cYT630), and slx5Δ (1dYT631) strains expressing GFP-Smt3p as in (A) were incubated at 32°C (semi-permissive for ubc9-1) for 5 h and imaged live. The insert shows gradient-like distribution of SUMO typical for wild type, which is noticeable at higher magnifications. Scale bars here and elsewhere are 5 µm. (C) SUMO conjugates are concentrated in the nucleolus in slx8Δ cells. The wild type (1014-YPH499) and slx8Δ (1aYT629) strains co-expressing GFP-Smt3p and Nop1p-mRFP were incubated at 30°C; cell images were captured live.
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Related In: Results  -  Collection

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pgen-1000215-g001: Smt3p conjugates are enriched in the nucleolus.(A) Alternative tagging of Smt3p expressed at the native levels enables the identification of sumoylated proteins. Total sumoylated proteins were purified by IMAC from the strains expressing wild-type levels of poly-His/FLAG-tagged Smt3p (HF-Smt3, 924-YPH499b), poly-His/FLAG/S-tag-Smt3p (HFS-Smt3, 1008-YPH499), and poly-His/FLAG/GFP-Smt3p (HFG-Smt3, 1014-YPH499. Sumoylated proteins here and thereafter are separated by PAGE and detected by Western-blotting using the anti-FLAG antibody. Arrows indicate the proportional size shifts between the free SUMO forms. Molecular weight markers (×1000) are shown on the left. (B) GFP-SUMO localization as a function of conjugation/de-conjugation. The wild type (1014-YPH499b), ubc9-1 (1cYT630), and slx5Δ (1dYT631) strains expressing GFP-Smt3p as in (A) were incubated at 32°C (semi-permissive for ubc9-1) for 5 h and imaged live. The insert shows gradient-like distribution of SUMO typical for wild type, which is noticeable at higher magnifications. Scale bars here and elsewhere are 5 µm. (C) SUMO conjugates are concentrated in the nucleolus in slx8Δ cells. The wild type (1014-YPH499) and slx8Δ (1aYT629) strains co-expressing GFP-Smt3p and Nop1p-mRFP were incubated at 30°C; cell images were captured live.
Mentions: We recently showed that the bulk of sumoylated proteins are concentrated in a subnucleolar area reminiscent of rDNA chromatin, if the nuclear desumoylation enzyme Smt4p is inactivated by the smt4 gene deletion [11]. This observation suggested that the SUMO pathway may play a major role in the nucleolus. Therefore, we closely examined strains expressing GFP-Smt3p under the native SMT3 promoter, as a sole source of SUMO. To monitor the GFP-Smt3p (HFG-Smt3) modification biochemically we also generated a shorter fusion (with the extended S-tag, HFS-Smt3). Similar patterns of conjugated protein bands were observed for both strains, except bands shifted accordingly to the tag size (Figure 1A). Both the HFS-Smt3 and HFG-Smt3 strains had the wild type doubling time (not shown), indicating that HFS-Smt3p and HFG-Smt3p fulfill key functions of SUMO.

Bottom Line: Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants.Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p.In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA.

ABSTRACT
SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

Show MeSH