Abstract
SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved ββαββαβ fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.
Highlights
Post-translational modification is an efficient and rapid way of controlling the activity of proteins
We show that SUMO/Pmt3 is phosphorylated on serine residues at its extreme N-terminus and that inability to phosphorylate SUMO/Pmt3 results in reduced levels of total SUMO/Pmt3 and reduced levels of high Mr SUMOcontaining species in vivo
S. pombe SUMO/Pmt3 is phosphorylated As part of our investigation into sumoylated species in S. pombe we undertook 2D PAGE
Summary
Post-translational modification is an efficient and rapid way of controlling the activity of proteins. SUMO is a member of the Ubl (ubiquitin-like) family of posttranslational modifiers. It has only 18% sequence identity with ubiquitin, its structure resembles that of ubiquitin, in that it contains the conserved ubiquitin bbabbab fold [3,4]. Ubiquitin has important roles in modifying the function of individual proteins required for specific processes, e.g. ubiquitination of PCNA (proliferating cell nuclear antigen) is required for bypass of replication blocking lesions in DNA [6,7,8]. SUMO modification has a variety of cellular functions, including roles in transcription, DNA damage responses, the cell cycle and nuclear transport e.g. It has been shown to be required for STUbL- (SUMO-targeted ubiquitin ligase)-dependent ubiquitination of target proteins e.g. [12,13,14]
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