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The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, we found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains. We discuss implications of this finding for function of other ubiquitin ligases.

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One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless K0 Cdc34 ⌬C is indistinguishable from Cdc34 ⌬C in ubiquitination of the prototype SCF Cdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0 cdc34 ⌬C or the cdc34 ⌬C allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.Posttranslational modification of proteins with ubiquitin has emerged as a major regulatory mechanism in eukaryotic cells, with both proteolysis-related and non-proteolysis-related functions (14,19,34,35). The variety of mechanisms of substrate recognition by specific ubiquitin ligases well reflects the large number of proteins targeted for ubiquitination. However, once a substrate is selected, its ubiquitination is catalyzed via a conserved cascade of ubiquitin transfer reactions.The first two steps of the ubiquitin transfer cascade do not involve a specific substrate protein and appear to be facilitated in a constitutive manner. In the first step, E1, called a ubiquitin-activating enzyme, forms a ubiquitin adenylate and subsequently a high-energy thiol ester bond with a carboxyl group of ubiquitin, thus activating it for nucleophilic attack. In the second step, the activated ubiquitin is trans-esterified to the active-site cysteine of one of several E2 ubiquitin-conjugating enzymes. The diversity in the ubiquitin transfer cascade begins when the ubiquitin-charged E2 is recruited to the proximity of a substrate through an interaction with a specific E3, which can be, but is not always, an enzyme. In the case of HECT (homology to E6-AP C terminus)-type E3s, E2 transfers ubiquitin to the catalytic site of E3 and E3 catalyzes ubiquitination. In contrast, in the case of RING (really interesting new gene)-type E3s, which do not have their own catalytic sites, E2 is directly responsible for ubiquitination of the E3-bound substrate. It is still unknown why such a difference evolved and why the enzymatic activity of HECT-type E3s is required if RING-type E3s can depend on the activity of E2 without having their own catalytic sites.

The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.Electronic supplementary materialThe online version of this article (10.1007/s11248-018-0092-z) contains supplementary material, which is available to authorized users.