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The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity.

Li SJ, Hochstrasser M - J. Cell Biol. (2003)

Bottom Line: Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase.Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others.These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.

ABSTRACT
Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A approximately 200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

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Subcellular localization of Ulp1 deletion derivatives. Localization of MYC9-tagged Ulp1 proteins by indirect immunofluorescence. Strains and plasmids were the same as in the legend to Fig. 2 D. Nuclei were stained with DAPI.
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fig5: Subcellular localization of Ulp1 deletion derivatives. Localization of MYC9-tagged Ulp1 proteins by indirect immunofluorescence. Strains and plasmids were the same as in the legend to Fig. 2 D. Nuclei were stained with DAPI.

Mentions: Full-length Ulp1 and ulp1-C478 proteins contained a consensus bipartite nuclear localization signal (NLS) and localized predominantly to the nuclear envelope, which was visible as a ring-like perinuclear fluorescence (Fig. 5). As seen previously, this rim staining was punctate (Li and Hochstrasser, 2000), which reflects Ulp1 binding to NPCs (Schwienhorst et al., 2000; Takahashi et al., 2000). The ulp1-C459 protein showed a detectable but less pronounced nuclear envelope localization, whereas ulp1-C449, which lacks the entire NLS, no longer concentrated clearly in a rim around the nucleus, although bright spots of stain on the perimeter of the nucleus were often seen. This transition from strong localization at NPCs to a more disperse distribution coincided with the strong increase in Smt3 conjugate accumulation seen with the same deletions (Fig. 2 C). Moreover, the ulp1-C275 and ulp1-C204 proteins no longer localized detectably at the nuclear rim (Fig. 5), and high levels of Smt3 conjugates accumulated in ulp1Δ cells expressing these derivatives (Fig. 2 C).


The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity.

Li SJ, Hochstrasser M - J. Cell Biol. (2003)

Subcellular localization of Ulp1 deletion derivatives. Localization of MYC9-tagged Ulp1 proteins by indirect immunofluorescence. Strains and plasmids were the same as in the legend to Fig. 2 D. Nuclei were stained with DAPI.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172760&req=5

fig5: Subcellular localization of Ulp1 deletion derivatives. Localization of MYC9-tagged Ulp1 proteins by indirect immunofluorescence. Strains and plasmids were the same as in the legend to Fig. 2 D. Nuclei were stained with DAPI.
Mentions: Full-length Ulp1 and ulp1-C478 proteins contained a consensus bipartite nuclear localization signal (NLS) and localized predominantly to the nuclear envelope, which was visible as a ring-like perinuclear fluorescence (Fig. 5). As seen previously, this rim staining was punctate (Li and Hochstrasser, 2000), which reflects Ulp1 binding to NPCs (Schwienhorst et al., 2000; Takahashi et al., 2000). The ulp1-C459 protein showed a detectable but less pronounced nuclear envelope localization, whereas ulp1-C449, which lacks the entire NLS, no longer concentrated clearly in a rim around the nucleus, although bright spots of stain on the perimeter of the nucleus were often seen. This transition from strong localization at NPCs to a more disperse distribution coincided with the strong increase in Smt3 conjugate accumulation seen with the same deletions (Fig. 2 C). Moreover, the ulp1-C275 and ulp1-C204 proteins no longer localized detectably at the nuclear rim (Fig. 5), and high levels of Smt3 conjugates accumulated in ulp1Δ cells expressing these derivatives (Fig. 2 C).

Bottom Line: Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase.Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others.These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.

ABSTRACT
Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A approximately 200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

Show MeSH
Related in: MedlinePlus