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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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The NH2-terminal domain of Ulp1 constrains its in vivo substrate specificity. (A) Smt3 protein conjugates in MHY1380 ulp2Δ cells carrying YCplac22-based plasmids with different ULP1 deletions or full-length ULP1 or ULP2. Cell lysates were fractionated by gradient SDS-PAGE. Positions of molecular mass markers are shown at left. The asterisks on the right indicate prominent sumoylated protein species observed in the ulp2Δ/vector lane but not in the lanes from cells transformed with certain ULP1 deletions (C459, C275, and/or C204). The double asterisk highlights a sumoylated protein found at higher levels upon expression of the above three ULP1 deletions. This might reflect incomplete removal of Smt3 from a substrate modified with multiple Smt3 molecules. (Bottom) The same filter reprobed with anti-PGK antibody. (B) Ulp1 NH2-terminal deletions can suppress ulp2Δ phenotypic defects. Strains used are the same as in A. For each strain, aliquots from 10-fold serial dilutions were spotted onto YPD plates (rich media) incubated at 30°C or 37°C and onto plates containing 0.1 M HU, or 20 μg/ml benomyl sulfate and incubated at 30°C for 5 d.
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fig6: The NH2-terminal domain of Ulp1 constrains its in vivo substrate specificity. (A) Smt3 protein conjugates in MHY1380 ulp2Δ cells carrying YCplac22-based plasmids with different ULP1 deletions or full-length ULP1 or ULP2. Cell lysates were fractionated by gradient SDS-PAGE. Positions of molecular mass markers are shown at left. The asterisks on the right indicate prominent sumoylated protein species observed in the ulp2Δ/vector lane but not in the lanes from cells transformed with certain ULP1 deletions (C459, C275, and/or C204). The double asterisk highlights a sumoylated protein found at higher levels upon expression of the above three ULP1 deletions. This might reflect incomplete removal of Smt3 from a substrate modified with multiple Smt3 molecules. (Bottom) The same filter reprobed with anti-PGK antibody. (B) Ulp1 NH2-terminal deletions can suppress ulp2Δ phenotypic defects. Strains used are the same as in A. For each strain, aliquots from 10-fold serial dilutions were spotted onto YPD plates (rich media) incubated at 30°C or 37°C and onto plates containing 0.1 M HU, or 20 μg/ml benomyl sulfate and incubated at 30°C for 5 d.

Mentions: When expressed at roughly endogenous levels, Ulp1 derivatives lacking all or some of the NH2-terminal noncatalytic domain failed to cleave many Smt3–protein conjugates efficiently (Fig. 2 C). This suggested a positive regulatory role(s) for the noncatalytic domain in substrate targeting. Remarkably, however, when we expressed these same NH2-terminally truncated versions of Ulp1 at low copy in strains lacking the other known desumoylating enzyme of yeast, Ulp2/Smt4, a substantial reduction in Smt3–protein conjugates was seen (Fig. 6 A). This was not true for full-length Ulp1 (or ulp1-C478) that was also expressed from a low copy plasmid. Most noticeably, the ulp1-C275 and ulp1–204 proteins eliminated the majority of the Smt3–protein species that characterize the ulp2Δ strain. Reduction of these sumoylated species correlated with substantial suppression of the ulp2Δ cellular phenotype, including temperature-sensitive growth and hypersensitivity to hydroxyurea (HU) or benomyl (Fig. 6 B). Growth on 0.1 M HU of ulp2Δ cells transformed with ulp1-C275 or ulp1-C204 was indistinguishable from the ULP2 transformants; ulp1-C204 was a slightly less effective suppressor of the high temperature and benomyl sensitivities. Catalytic activity was required inasmuch as the inactive ulp1-C173 protein failed to reduce the ulp2Δ-specific Smt3 conjugates or to restore growth under the tested conditions.


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

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

The NH2-terminal domain of Ulp1 constrains its in vivo substrate specificity. (A) Smt3 protein conjugates in MHY1380 ulp2Δ cells carrying YCplac22-based plasmids with different ULP1 deletions or full-length ULP1 or ULP2. Cell lysates were fractionated by gradient SDS-PAGE. Positions of molecular mass markers are shown at left. The asterisks on the right indicate prominent sumoylated protein species observed in the ulp2Δ/vector lane but not in the lanes from cells transformed with certain ULP1 deletions (C459, C275, and/or C204). The double asterisk highlights a sumoylated protein found at higher levels upon expression of the above three ULP1 deletions. This might reflect incomplete removal of Smt3 from a substrate modified with multiple Smt3 molecules. (Bottom) The same filter reprobed with anti-PGK antibody. (B) Ulp1 NH2-terminal deletions can suppress ulp2Δ phenotypic defects. Strains used are the same as in A. For each strain, aliquots from 10-fold serial dilutions were spotted onto YPD plates (rich media) incubated at 30°C or 37°C and onto plates containing 0.1 M HU, or 20 μg/ml benomyl sulfate and incubated at 30°C for 5 d.
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Related In: Results  -  Collection

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fig6: The NH2-terminal domain of Ulp1 constrains its in vivo substrate specificity. (A) Smt3 protein conjugates in MHY1380 ulp2Δ cells carrying YCplac22-based plasmids with different ULP1 deletions or full-length ULP1 or ULP2. Cell lysates were fractionated by gradient SDS-PAGE. Positions of molecular mass markers are shown at left. The asterisks on the right indicate prominent sumoylated protein species observed in the ulp2Δ/vector lane but not in the lanes from cells transformed with certain ULP1 deletions (C459, C275, and/or C204). The double asterisk highlights a sumoylated protein found at higher levels upon expression of the above three ULP1 deletions. This might reflect incomplete removal of Smt3 from a substrate modified with multiple Smt3 molecules. (Bottom) The same filter reprobed with anti-PGK antibody. (B) Ulp1 NH2-terminal deletions can suppress ulp2Δ phenotypic defects. Strains used are the same as in A. For each strain, aliquots from 10-fold serial dilutions were spotted onto YPD plates (rich media) incubated at 30°C or 37°C and onto plates containing 0.1 M HU, or 20 μg/ml benomyl sulfate and incubated at 30°C for 5 d.
Mentions: When expressed at roughly endogenous levels, Ulp1 derivatives lacking all or some of the NH2-terminal noncatalytic domain failed to cleave many Smt3–protein conjugates efficiently (Fig. 2 C). This suggested a positive regulatory role(s) for the noncatalytic domain in substrate targeting. Remarkably, however, when we expressed these same NH2-terminally truncated versions of Ulp1 at low copy in strains lacking the other known desumoylating enzyme of yeast, Ulp2/Smt4, a substantial reduction in Smt3–protein conjugates was seen (Fig. 6 A). This was not true for full-length Ulp1 (or ulp1-C478) that was also expressed from a low copy plasmid. Most noticeably, the ulp1-C275 and ulp1–204 proteins eliminated the majority of the Smt3–protein species that characterize the ulp2Δ strain. Reduction of these sumoylated species correlated with substantial suppression of the ulp2Δ cellular phenotype, including temperature-sensitive growth and hypersensitivity to hydroxyurea (HU) or benomyl (Fig. 6 B). Growth on 0.1 M HU of ulp2Δ cells transformed with ulp1-C275 or ulp1-C204 was indistinguishable from the ULP2 transformants; ulp1-C204 was a slightly less effective suppressor of the high temperature and benomyl sensitivities. Catalytic activity was required inasmuch as the inactive ulp1-C173 protein failed to reduce the ulp2Δ-specific Smt3 conjugates or to restore growth under the tested conditions.

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