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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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Contribution of the Ulp1 NH2 terminus to substrate recognition. (A) Overproduction of GST-Ulp1N417 and GST-Ulp1N255i leads to accumulation of sumoylated proteins. Strains and growth conditions as in B, but induction was for 6 h. Molecular mass markers are indicated. The vertical line denotes Smt3 protein conjugates. In the bottom panel, the filter was reprobed with anti-PGK antibody. (B) Overproduction of GST-Ulp1N417 inhibits Smt3 precursor processing. Mid-log phase cultures of wild-type MHY500 cells carrying pEMBL-GST-Ulp1N417, pGal4-ER-VP16 and pRS425-HA-Smt3-aty in selective medium were either induced with 100 nM β-estradiol (+N417) or not induced (–N417) for GST-Ulp1-N417 overproduction. After 4 h, the cells were collected by centrifugation and subjected to pulse–chase analysis. (C) Aliquots of 10-fold serially diluted cells carrying either empty vector (pEMBL-GST) or GAL1-driven alleles encoding Ulp1 NH2-terminal deletions were spotted to minimal plates containing glucose or galactose and incubated at 30°C for 4 d.
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fig3: Contribution of the Ulp1 NH2 terminus to substrate recognition. (A) Overproduction of GST-Ulp1N417 and GST-Ulp1N255i leads to accumulation of sumoylated proteins. Strains and growth conditions as in B, but induction was for 6 h. Molecular mass markers are indicated. The vertical line denotes Smt3 protein conjugates. In the bottom panel, the filter was reprobed with anti-PGK antibody. (B) Overproduction of GST-Ulp1N417 inhibits Smt3 precursor processing. Mid-log phase cultures of wild-type MHY500 cells carrying pEMBL-GST-Ulp1N417, pGal4-ER-VP16 and pRS425-HA-Smt3-aty in selective medium were either induced with 100 nM β-estradiol (+N417) or not induced (–N417) for GST-Ulp1-N417 overproduction. After 4 h, the cells were collected by centrifugation and subjected to pulse–chase analysis. (C) Aliquots of 10-fold serially diluted cells carrying either empty vector (pEMBL-GST) or GAL1-driven alleles encoding Ulp1 NH2-terminal deletions were spotted to minimal plates containing glucose or galactose and incubated at 30°C for 4 d.

Mentions: We assayed Smt3-cleaving activity of all the Ulp1 deletion proteins. The alleles were fused with the coding sequence for GST, and the fusion proteins were expressed in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimera was assayed by SDS-PAGE. Smt3 cleavage in vitro and growth complementation of ulp1Δ yeast correlated exactly (Fig. 1 and Fig. 2 A). Ulp1-C204 activity was lower than that of the full-length protein (Fig. 2 A). Kinetic analysis of His6-ubiquitin-Smt3-HA processing between the Smt3 and HA segments yielded values for vobs of 66 min−1 for GST-Ulp1 and 7.3 min−1 for GST-ulp1-C204. Processing in yeast cells of the natural COOH-terminal tripeptide Ala-Thr-Tyr from the Smt3 precursor was also tested (Fig. 2 B). This cleavage, which is due primarily to Ulp1 and not Ulp2 (Li and Hochstrasser, 2000), was slower in cells expressing ulp1-C204 than in those with full-length Ulp1 (Fig. 2 B and Fig. 3 B). On the other hand, Smt3 cleavage in vitro by all the other Ulp1 COOH-terminal fragments shown in Fig. 1 A was indistinguishable from cleavage by full-length Ulp1 with the possible exception of ulp1-C275, which was slightly slower (unpublished data). The activity of these fragments when overproduced in yeast also appeared to be comparable to that of full-length Ulp1 (see Fig. 4 A). In summary, the COOH-terminal catalytic domain of Ulp1 is both necessary and sufficient for the essential function of the protein in cell cycle progression and for Smt3 precursor cleavage.


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

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

Contribution of the Ulp1 NH2 terminus to substrate recognition. (A) Overproduction of GST-Ulp1N417 and GST-Ulp1N255i leads to accumulation of sumoylated proteins. Strains and growth conditions as in B, but induction was for 6 h. Molecular mass markers are indicated. The vertical line denotes Smt3 protein conjugates. In the bottom panel, the filter was reprobed with anti-PGK antibody. (B) Overproduction of GST-Ulp1N417 inhibits Smt3 precursor processing. Mid-log phase cultures of wild-type MHY500 cells carrying pEMBL-GST-Ulp1N417, pGal4-ER-VP16 and pRS425-HA-Smt3-aty in selective medium were either induced with 100 nM β-estradiol (+N417) or not induced (–N417) for GST-Ulp1-N417 overproduction. After 4 h, the cells were collected by centrifugation and subjected to pulse–chase analysis. (C) Aliquots of 10-fold serially diluted cells carrying either empty vector (pEMBL-GST) or GAL1-driven alleles encoding Ulp1 NH2-terminal deletions were spotted to minimal plates containing glucose or galactose and incubated at 30°C for 4 d.
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Related In: Results  -  Collection

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fig3: Contribution of the Ulp1 NH2 terminus to substrate recognition. (A) Overproduction of GST-Ulp1N417 and GST-Ulp1N255i leads to accumulation of sumoylated proteins. Strains and growth conditions as in B, but induction was for 6 h. Molecular mass markers are indicated. The vertical line denotes Smt3 protein conjugates. In the bottom panel, the filter was reprobed with anti-PGK antibody. (B) Overproduction of GST-Ulp1N417 inhibits Smt3 precursor processing. Mid-log phase cultures of wild-type MHY500 cells carrying pEMBL-GST-Ulp1N417, pGal4-ER-VP16 and pRS425-HA-Smt3-aty in selective medium were either induced with 100 nM β-estradiol (+N417) or not induced (–N417) for GST-Ulp1-N417 overproduction. After 4 h, the cells were collected by centrifugation and subjected to pulse–chase analysis. (C) Aliquots of 10-fold serially diluted cells carrying either empty vector (pEMBL-GST) or GAL1-driven alleles encoding Ulp1 NH2-terminal deletions were spotted to minimal plates containing glucose or galactose and incubated at 30°C for 4 d.
Mentions: We assayed Smt3-cleaving activity of all the Ulp1 deletion proteins. The alleles were fused with the coding sequence for GST, and the fusion proteins were expressed in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Processing of an 35S-labeled His6-ubiquitin-Smt3-HA chimera was assayed by SDS-PAGE. Smt3 cleavage in vitro and growth complementation of ulp1Δ yeast correlated exactly (Fig. 1 and Fig. 2 A). Ulp1-C204 activity was lower than that of the full-length protein (Fig. 2 A). Kinetic analysis of His6-ubiquitin-Smt3-HA processing between the Smt3 and HA segments yielded values for vobs of 66 min−1 for GST-Ulp1 and 7.3 min−1 for GST-ulp1-C204. Processing in yeast cells of the natural COOH-terminal tripeptide Ala-Thr-Tyr from the Smt3 precursor was also tested (Fig. 2 B). This cleavage, which is due primarily to Ulp1 and not Ulp2 (Li and Hochstrasser, 2000), was slower in cells expressing ulp1-C204 than in those with full-length Ulp1 (Fig. 2 B and Fig. 3 B). On the other hand, Smt3 cleavage in vitro by all the other Ulp1 COOH-terminal fragments shown in Fig. 1 A was indistinguishable from cleavage by full-length Ulp1 with the possible exception of ulp1-C275, which was slightly slower (unpublished data). The activity of these fragments when overproduced in yeast also appeared to be comparable to that of full-length Ulp1 (see Fig. 4 A). In summary, the COOH-terminal catalytic domain of Ulp1 is both necessary and sufficient for the essential function of the protein in cell cycle progression and for Smt3 precursor cleavage.

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