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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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Deletion analysis of the Ulp1 protein. (A) The set of terminal Ulp1 deletions used in the present work. Gray boxes represent the UD. Positions of the catalytic His514 and Cys580 residues and the C580S mutation are indicated. CC, potential coiled-coil region (aa 346–404). Catalytic activity of recombinant GST-Ulp1 deletion proteins purified from bacterial cells was determined by in vitro processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimeric substrate (see Fig. 2 A). (B) Complementation analysis of each deletion allele was done in a ulp1Δ strain by plasmid shuffling. Strain MHY1321 (ulp1Δ), which carries the wild-type ULP1 gene in a YCp50 (CEN, URA3) plasmid, was transformed with YCplac22 (TRP1)-based plasmids bearing the various ulp1 deletion alleles. Trp+ transformants were then streaked on 5-FOA plates and incubated at 30°C for 3 d to determine the ability of the different ULP1 deletions to support growth.
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fig1: Deletion analysis of the Ulp1 protein. (A) The set of terminal Ulp1 deletions used in the present work. Gray boxes represent the UD. Positions of the catalytic His514 and Cys580 residues and the C580S mutation are indicated. CC, potential coiled-coil region (aa 346–404). Catalytic activity of recombinant GST-Ulp1 deletion proteins purified from bacterial cells was determined by in vitro processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimeric substrate (see Fig. 2 A). (B) Complementation analysis of each deletion allele was done in a ulp1Δ strain by plasmid shuffling. Strain MHY1321 (ulp1Δ), which carries the wild-type ULP1 gene in a YCp50 (CEN, URA3) plasmid, was transformed with YCplac22 (TRP1)-based plasmids bearing the various ulp1 deletion alleles. Trp+ transformants were then streaked on 5-FOA plates and incubated at 30°C for 3 d to determine the ability of the different ULP1 deletions to support growth.

Mentions: Loss of Ulp1 is lethal in S. cerevisiae because the mutant cells are unable to traverse the G2/M phase of the cell cycle efficiently (Li and Hochstrasser, 1999). The Smt3-cleaving activity of the 621-residue Ulp1 enzyme is necessary for this essential in vivo function (Fig. 1). We asked whether any segments NH2-terminal to the catalytic domain (UD) could be deleted without loss of viability. A previous study had suggested that the UD by itself was insufficient for viability and, moreover, behaved as a dominant lethal (Mossessova and Lima, 2000). We were therefore surprised when we found that deletions could be extended as far as residue 417 (ulp1-C204) without a significant effect on growth rate at 30°C (Fig. 1). Mutant ulp1-C204 protein consists of the UD and little else. These experiments were performed with ulp1 alleles on low copy plasmids. When ulp1-C204 was integrated into the chromosome (at the LEU2 locus), viability was still seen although growth was slower than for wild-type cells, presumably because of lower ulp1-C204 expression levels (unpublished data). The differences in viability observed between studies appear to be due to properties of the specific constructs used (see Discussion).


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

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

Deletion analysis of the Ulp1 protein. (A) The set of terminal Ulp1 deletions used in the present work. Gray boxes represent the UD. Positions of the catalytic His514 and Cys580 residues and the C580S mutation are indicated. CC, potential coiled-coil region (aa 346–404). Catalytic activity of recombinant GST-Ulp1 deletion proteins purified from bacterial cells was determined by in vitro processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimeric substrate (see Fig. 2 A). (B) Complementation analysis of each deletion allele was done in a ulp1Δ strain by plasmid shuffling. Strain MHY1321 (ulp1Δ), which carries the wild-type ULP1 gene in a YCp50 (CEN, URA3) plasmid, was transformed with YCplac22 (TRP1)-based plasmids bearing the various ulp1 deletion alleles. Trp+ transformants were then streaked on 5-FOA plates and incubated at 30°C for 3 d to determine the ability of the different ULP1 deletions to support growth.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Deletion analysis of the Ulp1 protein. (A) The set of terminal Ulp1 deletions used in the present work. Gray boxes represent the UD. Positions of the catalytic His514 and Cys580 residues and the C580S mutation are indicated. CC, potential coiled-coil region (aa 346–404). Catalytic activity of recombinant GST-Ulp1 deletion proteins purified from bacterial cells was determined by in vitro processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimeric substrate (see Fig. 2 A). (B) Complementation analysis of each deletion allele was done in a ulp1Δ strain by plasmid shuffling. Strain MHY1321 (ulp1Δ), which carries the wild-type ULP1 gene in a YCp50 (CEN, URA3) plasmid, was transformed with YCplac22 (TRP1)-based plasmids bearing the various ulp1 deletion alleles. Trp+ transformants were then streaked on 5-FOA plates and incubated at 30°C for 3 d to determine the ability of the different ULP1 deletions to support growth.
Mentions: Loss of Ulp1 is lethal in S. cerevisiae because the mutant cells are unable to traverse the G2/M phase of the cell cycle efficiently (Li and Hochstrasser, 1999). The Smt3-cleaving activity of the 621-residue Ulp1 enzyme is necessary for this essential in vivo function (Fig. 1). We asked whether any segments NH2-terminal to the catalytic domain (UD) could be deleted without loss of viability. A previous study had suggested that the UD by itself was insufficient for viability and, moreover, behaved as a dominant lethal (Mossessova and Lima, 2000). We were therefore surprised when we found that deletions could be extended as far as residue 417 (ulp1-C204) without a significant effect on growth rate at 30°C (Fig. 1). Mutant ulp1-C204 protein consists of the UD and little else. These experiments were performed with ulp1 alleles on low copy plasmids. When ulp1-C204 was integrated into the chromosome (at the LEU2 locus), viability was still seen although growth was slower than for wild-type cells, presumably because of lower ulp1-C204 expression levels (unpublished data). The differences in viability observed between studies appear to be due to properties of the specific constructs used (see Discussion).

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