Biochemical characterization of the small ubiquitin-like modifiers of Chlamydomonas reinhardtii

Abstract

Dynamic modification of target proteins by small ubiquitin-like modifier (SUMO) is known to modulate many important cellular processes and is required for cell viability and development in all eukaryotes. However, understanding of SUMO systems in plants, especially in unicellular green algae, remains elusive. In this study, Chlamydomonas reinhardtii CrSUMO96, CrSUMO97 and CrSUMO148 were characterized. We show that the formation of polymeric CrSUMO96 and CrSUMO97 chains can be catalyzed either by the human SAE1/SAE2 and Ubc9 SUMOylation system in vitro or by an Escherichia coli chimeric SUMOylation system in vivo. An exposed C-terminal di-glycine motif of CrSUMO96 or CrSUMO97 is essential for functional SUMOylation. The human SUMO-specific protease, SENP1, demonstrates more processing activity for CrSUMO97 than for CrSUMO96. The CrSUMO148 precursor notably has four repeated di-glycine motifs at the C-terminus. This unique feature is not found in other known SUMO proteins. Interestingly, only 83-residual CrSUMO148(1-83) with the first di-glycine motif can form SAE1/SAE2-SUMO complex and further form polymeric chains with the help of Ubc9. More surprisingly, CrSUMO148 precursor is digested by SENP1, solely at the peptide bond after the first di-glycine motif although there are four theoretically identical processing sites in the primary sequence. This process directly generates 83-residual CrSUMO148(1-83) mature protein, which is exactly the form suitable for activation and conjugation. We also show that SENP1 displays similar isopeptidase activity in the deconjugation of polymeric CrSUMO96, CrSUMO97 or CrSUMO148 chains, revealing that the catalytic mechanisms of processing and deconjugation of CrSUMOs by SENP1 may differ.

Revised annotation and extended characterizations of components of the Chlamydomonas reinhardtii SUMOylation system

“…These Chlamydomonas SUMO proteins share the conserved diglycine residues that are required for processing by the SUMO proteases (Figure 2a). Similar to the previous report (Shin et al., 2010), SUMO1 contains four tandem IDAFVEQEGG repeats each with a potential diglycine cleavage site (Figure 2a). The tandem IDAFVEQEGG repeats, which seemed to be lacking in SUMO genes of Volvox carteri and Gonium pectoral , suggesting they are Chlamydomonas‐specific.…”

“…Consistent with the Phytozome annotation ( Chlamydomonas reinhardtii v5.5), two SUMO3 splice variants were isolated and verified by RT‐PCR. Similar to the previous study (Wang et al., 2008), all Chlamydomonas SUMO proteins including the newly identified SUMO3 revealed a conserved N‐terminal SUMOylation site (Lin et al., 2020) that may be important for polymerization as demonstrated in vitro (Shin et al., 2010) and suggested in vivo (Lin et al., 2020).…”

Arabidopsis SUMO E3 Ligase SIZ1 Is Involved in Excess Copper Tolerance

“…These steps require the participation of an E1-activating enzyme, an E2-conjugating enzyme (UBC9), and an E3 SUMO ligase to facilitate the transfer of SUMO from UBC9 to the acceptor Lys residue(s) in target proteins. Based on annotated databases and recent genetic and biochemical analyses, the components of sumoylation systems are also present in plants including algae (Chlamydomonas reinhardtii), dicots (Arabidopsis), and monocots (rice [Oryza sativa]; Colby et al, 2006;Miura et al, 2007;Nigam et al, 2008;Wang et al, 2008;Park et al, 2010;Shin et al, 2010). Sumoylation is involved in controlling cell growth and development (Miura and Hasegawa, 2010), embryogenesis (Colby et al, 2006;Saracco et al, 2007), and regulation of flowering time (Jin et al, 2008).…”

The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8

“…The strain number and culture condition of C. reinhardtii cells were described previously [39]. Briefly, C. reinhardtii cells were cultured at 25°C in the AC medium containing 10 mM NaNO 3 , 1 mM MgSO 4 , 0.1 mM CaCl 2 , 0.2 mM KH 2 PO 4 and 1.2 mM K 2 HPO 4 with sufficient air supply and under continuous lighting.…”