A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.

Claudio P Albuquerque,Kevin D Corbett,Ting Fu,Shawn Ma,Huilin Zhou,Eyan Yeung,Mariola J Edelmann

doi:10.1371/journal.pone.0143810

Claudio P Albuquerque, Kevin D Corbett + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0143810

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Abstract

A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.

Highlights

SUMO is a member of the ubiquitin-like protein family, and like ubiquitin, is covalently attached to target proteins via an isopeptide bond between the C-terminus of SUMO and the amino group of lysine on the target protein [1,2]
The SMT3 gene encodes SUMO in Saccharomyces cerevisiae and the Smt3 protein is activated by the heterodimeric Aos1-Uba2 complex, the SUMO E1 activating enzyme in S. cerevisiae [3]
Conventional MS-based methods to identify sumoylation sites rely on detection of a SUMO remnant (EQIGG, in S. cerevisiae) linked to sumoylation sites as a signature (Fig 1A, trypsin is used for illustration purpose)

Summary

Introduction

SUMO is a member of the ubiquitin-like protein family, and like ubiquitin, is covalently attached to target proteins via an isopeptide bond between the C-terminus of SUMO and the amino group of lysine on the target protein [1,2]. The SMT3 gene encodes SUMO in Saccharomyces cerevisiae and the Smt protein is activated by the heterodimeric Aos1-Uba complex, the SUMO E1 activating enzyme in S. cerevisiae [3]. The SUMO E3 ligases Siz, Siz and Mms are believed to control sumoylation of specific substrates in cells [5,6]. Like other post-translational modifications, protein sumoylation is reversible and two SUMO-specific isopeptidases, Ulp and Ulp, have been identified in S. cerevisiae [7,8]. While much is known about the identity and structure of the enzymes that catalyze reversible sumoylation [9], their regulation and substrate selectivity in vivo are not well understood

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