In Vitro Studies Reveal a Sequential Mode of Chain Processing by the Yeast SUMO (Small Ubiquitin-related Modifier)-specific Protease Ulp2

Eckhoff, Julia; Dohmen, R. Jürgen

doi:10.1074/jbc.m114.622217

Cited by 20 publications

(15 citation statements)

References 65 publications

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“…Thus, Ulp2's C-terminal SIM helps it to preferentially cleave longer SUMO chains, most likely via its increased binding affinity to longer SUMO chains. Our finding here is consistent with a prior study showing that Ulp2 preferentially cleaves longer SUMO chains in vitro (38). However, we have…”

Section: Sumo Binding Mediates Ulp2 Substrate Specificitysupporting

confidence: 94%

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease

Albuquerque

Suhandynata

Carlson

et al. 2018

Journal of Biological Chemistry

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Ulp1 and Ulp2, in the yeast , are the founding members of deSUMOylating enzymes. These enzymes removemall biquitin-likedifier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme. However, the mechanism for Ulp2's substrate specificity has been insufficiently understood. Here we show that the C-terminal regulatory domain of Ulp2 contains three distinct, yet conserved, motifs that control its substrate specificity and cell growth. Among them, a SUMO-interacting motif (SIM) was found to coordinate with the domain of Ulp2 that binds to the nucleolar protein Csm1 to ensure maximal deSUMOylation of Ulp2's nucleolar substrates. We found that whereas the Csm1-binding domain of Ulp2 recruits this enzyme to the nucleolus, Ulp2's C-terminal SIM promotes its SUMO protease activity and plays a key role in mediating the specificity of Ulp2. Thus, the substrate specificity of Ulp2 is controlled by both its subcellular localization and the SUMOylation status of its substrates. These findings illustrate the highly coordinated and dynamic nature of the SUMO pathways in maintaining homeostasis of intracellular SUMOylation.

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Section: Sumo Binding Mediates Ulp2 Substrate Specificitysupporting

confidence: 94%

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease

Albuquerque

Suhandynata

Carlson

et al. 2018

Journal of Biological Chemistry

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“…Large amounts of high molecular weight (HMW) SUMO conjugates, which accumulate in the stacker of SDS-PAGE gels, are observed in ulp2D cells. Correspondingly, Ulp2 has a preference in vitro for cleaving between SUMO monomers in polySUMO chains (Hickey et al 2012;Eckhoff and Dohmen 2015). The identities of most of the HMW-SUMO conjugates are unknown, as is their contribution to the growth defects of ulp2D cells (Bylebyl et al 2003).…”

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confidence: 99%

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Gillies

Hickey

et al. 2016

Genetics

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In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2D and ulp2D slx5D cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2D, slx5D, and ulp2D slx5D cells. The increase in sumoylation of Net1 and Tof2 in ulp2D, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2. Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2D cells and both factors largely regain this association in ulp2D slx5D. A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/ Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2D cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2D cells, which is also rescued by elimination of Slx5. Additionally, overexpression of Sir2, another RFBassociated factor, suppresses the growth defect of ulp2D cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2D cellular defects. KEYWORDS SUMO; Ulp2; rDNA; RENT complex; Fob1 P OST-TRANSLATIONAL protein modifications provide a common mechanism for regulating protein-protein interactions, protein localization, and protein degradation. The small ubiquitin-related modifier (SUMO) protein family modifies target proteins through covalent attachment to lysine side chains (Johnson 2004). The function of substrate "sumoylation" varies with the protein that is sumoylated, but a common role is to modulate the assembly of large protein complexes either by creating additional binding sites or by blocking existing sites (Kerscher 2007).Protein sumoylation occurs through an enzymatic cascade similar to ubiquitylation (Johnson 2004). In Saccharomyces cerevisiae, a single gene, SMT3, codes for SUMO, and as in most species, SUMO ligation is essential for viability. Removal of SUMO from target proteins is also an important regulatory step. S. cerevisiae has two SUMO proteases, Ulp1 and Ulp2 (Hickey et al. 2012). Most Ulp1 is bound to the nuclear pore complex (NPC), and the enzyme is essential for cell-cycle progression. NPC tethering regulates Ulp1 activity by restricting its access to sumoylated proteins (Li and Hochstrasser 2003;Panse et al. 2003). Ulp1 also processes the SUMO precursor by removing its last three residues, thereby exposing the C-terminal Gly-Gly motif required for SU...

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“…The in vitro results are well consistent with the in vivo observation ( Figure 3B), and thus eliminated the possibility of two-hybrid false positive. Combined, the results suggested by, 2006; Eckhoff & Dohmen, 2015;Park et al, 2011;Yang, Hsu, Ting, Liu, & Hwang, 2006). In addition, human SUMO2 and SUMO3 formed chains in vitro and in vivo, primarily through a conserved acceptor, Lys11 (Park et al, 2011;Tatham et al, 2001).…”

Section: Rin13 Was Sumoylated By Sumo1 2 3 and 5 In E Coli Sumoylamentioning

confidence: 64%

Characterization of The Interaction Between RIN13 (RPM1-Interacting13) and Sumo Proteins in Arabidopsis Thaliana

et al. 2017

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Small Ubiquitin-related MOdifier (SUMO) proteins can be found in many organisms, including A. thaliana, which possesses 9 SUMO genes. SUMO binds to various target proteins in a reversible reaction called SUMOylation. SUMOylation participates in transcription, chromosome organization, proteins localizations and stress responses. Our study showed that RIN13 (RPM1-Interacting13/At2g20310) is a target of SUMOylation, which was initially found by interaction between this protein and AtSCE1a (E2). Recent report showed that overexpression of RIN13 enhanced the resistance to pathogen without inducing hypersensitive response. However, the molecular interaction between RIN13 and SUMO proteins and its significance have not been studied yet. Thus, our study aimed to characterize the Interaction between RIN13 and SUMO proteins in A. thaliana. The result showed an isoform-specific SUMOylation between RIN13 and SUMO proteins. RIN13 is SUMOylated by SUMO1, 2, 3, and 5. Though expressed ubiquitously in A.thaliana, fluorescence microscopy showed that RIN13 localizes subcellularly in the nuclear body. Moreover, complete abolishment of SUMOylation with inactive E2 suggests the exclusion of RIN13 from nuclear body. These results showed that SUMOylation affected RIN13 localization, and indirectly influenced its interaction to other proteins and putative function. This paper presents evidence of RIN13 SUMOylation. Furthermore, RIN13 function in pathogenic resistance is shown to be supported by SUMOylation. Thus, this study enhanced the understanding of SUMO in plants and served as reference to molecular studies concerning post-translational modification of SUMO. How to Cite

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In Vitro Studies Reveal a Sequential Mode of Chain Processing by the Yeast SUMO (Small Ubiquitin-related Modifier)-specific Protease Ulp2

Cited by 20 publications

References 65 publications

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Characterization of The Interaction Between RIN13 (RPM1-Interacting13) and Sumo Proteins in Arabidopsis Thaliana

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