title: A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.
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.
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Claudio P
authors.initials: CP
authors.lastname: Albuquerque
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Eyan
authors.initials: E
authors.lastname: Yeung
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Shawn
authors.initials: S
authors.lastname: Ma
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Ting
authors.initials: T
authors.lastname: Fu
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.|Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Corbett
authors.affiliation: Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, California, United States of America.|Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.|Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Huilin
authors.initials: H
authors.lastname: Zhou
mesh_terms: D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin