Positively charged amino acids flanking a sumoylation consensus tetramer on the 110 kDa tri-snRNP component SART1 enhance sumoylation efficiency

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the epsilon-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110 kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys(94) and Lys(141) of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys(94) and Lys(141), but not Lys(709) and Lys(742), resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys(94). These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites. (C) 2010 Elsevier B.V. All rights reserved.