Abstract
Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.
Highlights
The majority of short-lived cellular proteins are targeted for degradation by the 26 S proteasome in response to assembly of degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues [1]
The dependence of initial rate for E2-ubiquitin thiolester formation versus HsUbc2b concentration can be used to examine the kinetics of ubiquitin transfer in the key step linking the half-reactions of ubiquitin activation and ligation under conditions for which [E1]o ϽϽ [HsUbc2b]o [35]
Of that determined from a parallel incubation for which E1 and HsUbc2b were present at equivalent concentrations of 1 M, whereas at 0.7 nM activating enzyme the end point for HsUbc2b thiolester formation was 45% of that predicted
Summary
The majority of short-lived cellular proteins are targeted for degradation by the 26 S proteasome in response to assembly of degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues [1]. Because each ligase family recognizes a cognate E2 or its orthologs, rates of conjugation through the different pathways and, their contribution to degradative targeting, depend on efficient loading of these enzymes by the E1 ternary complex [9]. We extend these studies to protein-protein interactions among the components of the Nend rule targeting pathway for ubiquitin conjugation This pathway requires the E3 isozyme Ubr in Saccharomyces cerevisiae [2] and the 19.7-kDa E2 isozyme Rad6/ScUbc22 [25]. Activity assays involving 125I-ubiquitin thiolester formation have been exploited to quantitate intracellular concentrations of E1 and Ubc within selected cell lines Comparison of these binding constants with the concentrations of the components required for N-end ruledependent ligation suggests that the three enzymes of this pathway minimally form dynamic binary complexes within the cell
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
