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, E220K and E232K, 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 E220K 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 E220K 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 kcat (1.9 min-1) and Km (20 μM). A slow rate of multiubiquitination involving formation of extended ubiquitin homopolymers on the histones was also observed with RAD6 and E220K Comparison of conjugate patterns among native, reductively methylated, and K48R ubiquitin variants demonstrated that the linkage between ubiquitin moieties formed by E220K 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, E232K, in showing a specificity for conjugation to bovine serum albumin rather than to core histones. Both CDC34 and E232K exhibit a marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin. Calculations based on a model ubiquitin conjugation reaction indicated that E232K and CDC34 preferentially catalyzed multiubiquitination over ligation of the polypeptide directly to target proteins. Formation of such multiubiquitin homopolymers by E232K and CDC34 suggests these enzymes may commit their respective target proteins to degradation via an E3-independent pathway.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - Mar 15 1991|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology