Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products: Comparison to their putative rabbit homologs, E2_20K and E2_32K

The recombinant yeast RAD6 and CDC34 gene products were expressed in Escherichia coli extracts and purified to apparent homogeneity. The physical and catalytic properties of RAD6 and CDC34 were similar but distinct from their putative rabbit reticulocyte homologs, E2_20K and E2_32K, respectively. Like their reticulocyte counterparts, RAD6 and CDC34 are bifunctional enzymes competent in both ubiquitin:protein ligase (E3)-independent and E3-dependent conjugation reactions. RAD6 and E2_20K exhibit marked specificity for the conjugation of core histones and catalyze the processive ligation of up to three ubiquitin moieties directly to such model substrates. RAD6 differed from its putative E2_20K homolog in exhibiting simple saturation behavior in the kinetics of histone conjugation and in being unable to distinguish kinetically between core histones H2A and H2B, yielding identical values of k_cat (1.9 min^-1) and K_m (20 μM). A slow rate of multiubiquitination involving formation of extended ubiquitin homopolymers on the histones was also observed with RAD6 and E2_20K Comparison of conjugate patterns among native, reductively methylated, and K48R ubiquitin variants demonstrated that the linkage between ubiquitin moieties formed by E2_20K and RAD6 was not through Lys-48 of ubiquitin, the site previously demonstrated as a strong signal for degradation of the target protein. In contrast, CDC34 differs from its putative homolog, E2_32K, in showing a specificity for conjugation to bovine serum albumin rather than to core histones. Both CDC34 and E2_32K exhibit a marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin. Calculations based on a model ubiquitin conjugation reaction indicated that E2_32K and CDC34 preferentially catalyzed multiubiquitination over ligation of the polypeptide directly to target proteins. Formation of such multiubiquitin homopolymers by E2_32K and CDC34 suggests these enzymes may commit their respective target proteins to degradation via an E3-independent pathway.