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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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Comparison of Ulp1 catalytic activity in vitro and function in vivo. (A) Enzymatic activity of selected GST-Ulp1 derivatives. Cleavage of purified 35S-labeled His6-ubiquitin-Smt3-HA by purified GST fusions was assayed at 30°C for 30 min and stopped by addition of SDS-loading buffer. (B) In vivo processing of HA-Smt3-aty. MHY1321 cells carrying pRS425-HA-Smt3-aty and either wild-type ULP1 or ulp1-C204 in YCplac22 were pulse labeled with 35S-Translabel for 5 min and chased in buffer containing cycloheximide and an excess of unlabeled methionine. At the indicated time points, aliquots of cells were withdrawn and processed for immunoprecipitation with anti-HA antibodies. The positions of precursor HA-Smt3-aty and the processed product are indicated. Because of the efficiency of processing, it is often difficult to detect precursor in ULP1 cells at the initial time point. (C) Smt3 protein profiles of ulp1Δ (MHY1321) cells carrying the indicated plasmids with different Ulp1 deletions. Cell extracts were fractionated through an 11–14% gradient SDS gel and assayed by immunoblotting with a polyclonal anti-Smt3 antiserum. The membrane was reprobed with anti-PGK (phosphoglycerate kinase) to compare the protein loading (bottom). Positions of marker proteins (kD) are indicated on the left. (D) Anti-MYC immunoblot showing expression levels of MYC9-tagged Ulp1 derivatives. ULP1 alleles used the ULP1 promoter and were based in YCplac22. COOH-terminally MYC9-tagged Ulp1, C478, C459, C449, C275, and C204 proteins, which were indistinguishable in growth from the untagged alleles use in C, were expressed in strain MHY1321 (ulp1Δ). The recessive lethal alleles encoding the catalytically inactive Ulp1-N417-MYC9 and Ulp1-C173-MYC9 proteins were expressed in congenic wild-type MHY500 cells. (Bottom) The same membrane reprobed with anti-PGK antibody.
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fig2: Comparison of Ulp1 catalytic activity in vitro and function in vivo. (A) Enzymatic activity of selected GST-Ulp1 derivatives. Cleavage of purified 35S-labeled His6-ubiquitin-Smt3-HA by purified GST fusions was assayed at 30°C for 30 min and stopped by addition of SDS-loading buffer. (B) In vivo processing of HA-Smt3-aty. MHY1321 cells carrying pRS425-HA-Smt3-aty and either wild-type ULP1 or ulp1-C204 in YCplac22 were pulse labeled with 35S-Translabel for 5 min and chased in buffer containing cycloheximide and an excess of unlabeled methionine. At the indicated time points, aliquots of cells were withdrawn and processed for immunoprecipitation with anti-HA antibodies. The positions of precursor HA-Smt3-aty and the processed product are indicated. Because of the efficiency of processing, it is often difficult to detect precursor in ULP1 cells at the initial time point. (C) Smt3 protein profiles of ulp1Δ (MHY1321) cells carrying the indicated plasmids with different Ulp1 deletions. Cell extracts were fractionated through an 11–14% gradient SDS gel and assayed by immunoblotting with a polyclonal anti-Smt3 antiserum. The membrane was reprobed with anti-PGK (phosphoglycerate kinase) to compare the protein loading (bottom). Positions of marker proteins (kD) are indicated on the left. (D) Anti-MYC immunoblot showing expression levels of MYC9-tagged Ulp1 derivatives. ULP1 alleles used the ULP1 promoter and were based in YCplac22. COOH-terminally MYC9-tagged Ulp1, C478, C459, C449, C275, and C204 proteins, which were indistinguishable in growth from the untagged alleles use in C, were expressed in strain MHY1321 (ulp1Δ). The recessive lethal alleles encoding the catalytically inactive Ulp1-N417-MYC9 and Ulp1-C173-MYC9 proteins were expressed in congenic wild-type MHY500 cells. (Bottom) The same membrane reprobed with anti-PGK antibody.

Mentions: 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.


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

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

Comparison of Ulp1 catalytic activity in vitro and function in vivo. (A) Enzymatic activity of selected GST-Ulp1 derivatives. Cleavage of purified 35S-labeled His6-ubiquitin-Smt3-HA by purified GST fusions was assayed at 30°C for 30 min and stopped by addition of SDS-loading buffer. (B) In vivo processing of HA-Smt3-aty. MHY1321 cells carrying pRS425-HA-Smt3-aty and either wild-type ULP1 or ulp1-C204 in YCplac22 were pulse labeled with 35S-Translabel for 5 min and chased in buffer containing cycloheximide and an excess of unlabeled methionine. At the indicated time points, aliquots of cells were withdrawn and processed for immunoprecipitation with anti-HA antibodies. The positions of precursor HA-Smt3-aty and the processed product are indicated. Because of the efficiency of processing, it is often difficult to detect precursor in ULP1 cells at the initial time point. (C) Smt3 protein profiles of ulp1Δ (MHY1321) cells carrying the indicated plasmids with different Ulp1 deletions. Cell extracts were fractionated through an 11–14% gradient SDS gel and assayed by immunoblotting with a polyclonal anti-Smt3 antiserum. The membrane was reprobed with anti-PGK (phosphoglycerate kinase) to compare the protein loading (bottom). Positions of marker proteins (kD) are indicated on the left. (D) Anti-MYC immunoblot showing expression levels of MYC9-tagged Ulp1 derivatives. ULP1 alleles used the ULP1 promoter and were based in YCplac22. COOH-terminally MYC9-tagged Ulp1, C478, C459, C449, C275, and C204 proteins, which were indistinguishable in growth from the untagged alleles use in C, were expressed in strain MHY1321 (ulp1Δ). The recessive lethal alleles encoding the catalytically inactive Ulp1-N417-MYC9 and Ulp1-C173-MYC9 proteins were expressed in congenic wild-type MHY500 cells. (Bottom) The same membrane reprobed with anti-PGK antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172760&req=5

fig2: Comparison of Ulp1 catalytic activity in vitro and function in vivo. (A) Enzymatic activity of selected GST-Ulp1 derivatives. Cleavage of purified 35S-labeled His6-ubiquitin-Smt3-HA by purified GST fusions was assayed at 30°C for 30 min and stopped by addition of SDS-loading buffer. (B) In vivo processing of HA-Smt3-aty. MHY1321 cells carrying pRS425-HA-Smt3-aty and either wild-type ULP1 or ulp1-C204 in YCplac22 were pulse labeled with 35S-Translabel for 5 min and chased in buffer containing cycloheximide and an excess of unlabeled methionine. At the indicated time points, aliquots of cells were withdrawn and processed for immunoprecipitation with anti-HA antibodies. The positions of precursor HA-Smt3-aty and the processed product are indicated. Because of the efficiency of processing, it is often difficult to detect precursor in ULP1 cells at the initial time point. (C) Smt3 protein profiles of ulp1Δ (MHY1321) cells carrying the indicated plasmids with different Ulp1 deletions. Cell extracts were fractionated through an 11–14% gradient SDS gel and assayed by immunoblotting with a polyclonal anti-Smt3 antiserum. The membrane was reprobed with anti-PGK (phosphoglycerate kinase) to compare the protein loading (bottom). Positions of marker proteins (kD) are indicated on the left. (D) Anti-MYC immunoblot showing expression levels of MYC9-tagged Ulp1 derivatives. ULP1 alleles used the ULP1 promoter and were based in YCplac22. COOH-terminally MYC9-tagged Ulp1, C478, C459, C449, C275, and C204 proteins, which were indistinguishable in growth from the untagged alleles use in C, were expressed in strain MHY1321 (ulp1Δ). The recessive lethal alleles encoding the catalytically inactive Ulp1-N417-MYC9 and Ulp1-C173-MYC9 proteins were expressed in congenic wild-type MHY500 cells. (Bottom) The same membrane reprobed with anti-PGK antibody.
Mentions: 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.

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