![]() UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. ![]() Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases.
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