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  • Taken together these assays demonstrate that SUMOD positioni


    Taken together, these assays demonstrate that SUMOD positioning is essential for all E3 ligases but dispensable for E2 and S*E2 sumoylation reactions. Thus, we propose SUMOD positioning as a key criterion to describe the enzymatic function of SUMO E3 ligases that distinguishes them from other enhancing activities like cofactors or the S*E2.
    Summary and conclusions In vitro sumoylation assays offer powerful tools to study sumoylation. They can be used to address several aspects, such as identification and analysis of SUMO substrates, mapping their specific sumoylation sites or determining their specific conjugating and deconjugating enzymes. In addition, these assays allow the functional characterization of the FTY720 Phosphate themselves, like which surfaces are important for certain functions, including E1–E2, E2–substrate, E3–substrate, and E2–E3 interactions. They further permit conclusions about the stoichiometric ratio between the substrate and the enzymes required for efficient substrate modification. A stoichiometric modifier/substrate ratio rather describes a cofactor, while potent activity at substoichiometric ratios points to what we define as E3 ligases. However, there can be some ambiguity in such an assignment as we demonstrate for FTY720 Phosphate S*E2. S*E2 definitely shows enhanced catalytic activation compared to the unmodified E2 and is activated in a substrate-specific manner. However, the enhancing role of the additional SUMO is better described as a cofactor function that stabilizes the interaction of the substrate with the E2, and no additional catalytic activity appears to be involved (Knipscheer et al., 2008). Thus, it is important to also investigate other characteristics of SUMO E3 ligases, like the dependence of a scaffold SUMOB binding to the backside of the E2 that is important for all known SUMO E3 ligases except for RanBP2. Ultimately, the most conclusive assay to demonstrate SUMO E3 ligase activity is by demonstrating dependence of donor SUMOD positioning for the discharge of the E2 and the SUMOD transfer to the substrate.
    Introduction Ubiquitination is a well-characterized post-translational modification that is critical for regulating a wide range of cellular processes in eukaryotic organisms, including higher plants [1], [2], [3]. The conjugation of ubiquitin (Ub) to target proteins is sequentially carried out by E1 Ub-activating enzymes, E2 Ub-conjugating enzymes, and E3 Ub-ligases [4], [5], [6]. In plant genomes, there are few E1s, approximately 40 E2s, and more than 1000 E3s [7], [8]. With their abundance and target-binding activities, E3s generally determine the specificity of the ubiquitination pathway. In humans, however, E2s not only interact with E1s and E3s to receive and transfer Ub, respectively, but also regulate, at least in part, the length and topology of the poly-Ub chain and the efficiency of poly-ubiquitination conducted by E3s [5], [9]. Thus, E2s are not simply involved in ubiquitination, but are one of the regulators in the ubiquitination pathway. E3s contain one of three distinct functional domains: HECT, RING, or U-box [10], [11], [12]. The U-box motif shares a similar structure with the RING domain, but does not bind zinc ions to act as an E3 Ub-ligase [11], [12]. Compared to humans and yeast, higher plants have a large number of U-box motif-containing E3 Ub-ligases. Arabidopsis, a dicot model plant, contains 64 U-box genes [13] and the monocot model crop rice possesses at least 77 U-box genes [14]. Based on their primary sequences and presence of specific domains, the U-box E3s are divided into nine different classes [14]. Class II and III U-box E3s are the most abundant E3s and are typified by the presence of a protein–protein interacting armadillo (ARM) repeat domain [13], [14], [15]. Plant U-box proteins have a role in diverse plant-specific phenomena, including hormone signaling [16], [17], responses to abiotic/biotic stress [18], [19], [20], self-incompatibility [21], and flowering time control [22]. For instance, rice ARM-U-box E3 SPL11 negatively regulates programmed cell death [20]. SPL11 plays an additional role in regulating flowering time by mono-ubiquitinating its target protein SPIN1 that represses flowering by down-regulating the flowering promoter gene HD3A[22]. OsPUB15, which encodes a class II ARM-U-box E3, regulates oxidative stress and cell death responses by reducing reactive oxygen species in rice [19].