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Mentions: The two yeast SUMO isopeptidases, Ulp1 and Ulp2, have distinct in vivo substrate specificities, and the data presented here provide several important clues about the molecular underpinnings of these differences. Based on both the present results (summarized in Fig. 7) and our earlier work, it is clear that Ulp1 has the ability to cleave a very broad range of sumoylated substrates in vitro. However, at its normal levels of expression in vivo the spectrum of proteins acted upon by Ulp1 is more limited. A critical element in controlling Ulp1 activity appears to be its restricted localization: high levels are normally only found at the nuclear envelope (NPC), and loss of this constraint correlates with the ability to act on additional sumoylated proteins. On the other hand, a failure to localize to NPCs also may impair Ulp1 action toward many of its normal targets, suggesting a specific requirement for Ulp1 at the NPC. The noncatalytic domain of Ulp1 may also control targeting to at least a subset of substrates by means other than NPC binding. Surprisingly, the essential function of Ulp1 in cell cycle progression does not require the noncatalytic domain, indicating that certain substrates can be recognized by the catalytic domain alone. We discuss the implications of these data for both the evolution of multiple SUMO isopeptidases with diverse specificities and the potential relationship between the yeast Ulps and those from other eukaryotes.
The Ulp1 SUMO isopeptidase
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.
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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