Systematic Identification and Analysis of Mammalian Small Ubiquitin-like Modifier Substrates

Christian B. Gocke,Jungseog Kang,Hongtao Yu

doi:10.1074/jbc.m411718200

Christian B. Gocke, Jungseog Kang + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m411718200

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Abstract

Small ubiquitin-like modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an in vitro expression cloning approach, we have identified 40 human SUMO1 substrates. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. We have validated the sumoylation of 24 substrates in living cells. Analysis of this panel of SUMO substrates leads to the following observations. 1) Sumoylation is more efficient in vitro than in living cells. Polysumoylation occurs on several substrates in vitro. 2) SUMO isopeptidases have little substrate specificity. 3) The SUMO ligases, PIAS1 and PIASxbeta, have broader substrate specificities than does PIASy. 4) Although SUMO1 and SUMO2 are equally efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo. 5) Most SUMO substrates localize to the nucleus, and sumoylation does not generally affect their subcellular localization. Therefore, sumoylation appears to regulate the functions of its substrates through multiple, context-dependent mechanisms.

Highlights

Covalent conjugation of SUMO1 is an important post-translational modification that regulates protein functions in eukaryotes [1,2,3,4,5,6,7]
3) The Small ubiquitin-like modifier (SUMO) ligases, PIAS1 and PIASx␤, have broader substrate specificities than does PIASy. 4) SUMO1 and SUMO2 are efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo
We have confirmed the sumoylation of 24 substrates in living cells. Using this panel of substrates, we have investigated the extent of polysumoylation of substrates, the conjugation selectivity of SUMO1 and SUMO2, and the specificities of SUMO isopeptidases and ligases

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The coding regions of SUMO1 [1–97], SUMO2, PIASy, SENP1, SENP2, and SENP3 were amplified from human fetal thymus cDNA library (BD Biosciences) by PCR. Ubc was cloned into a pCS2 vector containing a C-terminal FLAG tag. Protein Expression and Purification—DNA fragments encoding the wild-type or KØ mutant of His6-SUMO1 and the wild-type His6-SUMO2 were subcloned into pET28a These proteins were expressed in BL21(DE3) and purified using nickel-nitrilotriacetic acid beads per the manufacturer’s protocols (Qiagen). 1 ␮l of DNA was in vitro transcribed and translated in reticulocyte lysate in the presence of [35S]methionine and subjected to in vitro sumoylation reactions, which contained 2 ␮l of in vitro transcribed and translated product, 2 ␮g of Aos1-Uba2, 0.5 ␮g of Ubc9, 1 ␮g of SUMO1, and 1 ␮l of energy mix (150 mM phosphocreatine, 20 mM ATP, 2 mM EGTA, 20 mM MgCl2, adjusted pH to 7.7). Images were acquired using the Intelligent Imaging software and pseudo-colored in Adobe Photoshop

RESULTS

Identification of Mammalian SUMO Substrates

Cytosol ϩ

Efficiency in vitro

DISCUSSION