Protein interactions within the N-end rule ubiquitin ligation pathway.
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.
Siepmann
Thomas J
TJ
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Bohnsack
Richard N
RN
Tokgöz
Zeynep
Z
Baboshina
Olga V
OV
Haas
Arthur L
AL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D018938:Caco-2 Cells; D002384:Catalysis; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
D004952:Esters; D013438:Sulfhydryl Compounds; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases