Biochemical characterization of the small ubiquitin-like modifiers of Chlamydomonas reinhardtii
Date Issued
2011
Date
2011
Author(s)
Shin, Yung-Cheng
Abstract
SUMO (small ubiquitin-like modifier) was originally discovered in 1996, as a peptide associating with RanGAP1 (Ran GTPase-activating protein 1). More studies subsequently found that SUMO can modify many proteins and influence many cellular processes of gene expression and signal transduction in eukaryotes. However, investigation of sumoylation on plant systems, especially on unicellular green algae, remains little. Thus, this work was aimed to study the SUMO system in Chlamydomonas reinhardtii and also characterize their biochemical properties. Three genes encoding for SUMO proteins from Chlamydomonas reinhardtii have been identified and named CrSUMO96, CrSUMO97 and CrSUMO148. Using the E1 activating enzyme (SAE1/SAE2 or Aos1/Uba2) and E2 conjugating enzyme (Ubc9) of the animal SUMO system, the present data reveal that CrSUMO96 and CrSUMO97 with an exposed di-glycine motif at the C-terminus can form polymeric chains, whereas the polymeric chain was not observed by using the full length of CrSUMO96 or CrSUMO97 as the assay substrate. It clearly demonstrates that the exposed C-terminal di-glycine end is required for sumoylation. In SUMO processing activity assay, human SENP1 showed greater processing activity toward CrSUMO97 than CrSUMO96. Interestingly, CrSUMO148 have four repeated di-glycine motif at the C-terminus, which are not found in other SUMO proteins. SENP1 specifically digests CrSUMO148 at the first di-glycine motif to generate CrSUMO1481-83. Furthermore, only CrSUMO1481-83 can form polymeric chain in the in vitro sumoylation assay. The deconjugation activity of SENP1 towards poly-SUMO chains showed that all polyCrSUMO chains can be completely deconjugated to form SUMO monomers.
Subjects
SUMO
SENP1
di-glycine motif
SAE1/SAE2
Aos1/Uba2
Ubc9
Type
thesis
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