The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes.

Abstract

The covalent ligation of the 8.6-kDa polypeptide ubiquitin to various cellular target proteins is believed to represent a fundamental regulatory process. In this mechanism, the ATP-coupled activation and subsequent ligation of ubiquitin are catalyzed by separate enzymes (E1 and E3, respectively) functionally linked by ubiquitin carrier protein (E2). Carrier protein has been proposed to constitute a family of isozymes having molecular masses of 14, 17, 20, 24, and 32 kDa whose role is to shuttle activated polypeptide in the form of a high-energy thiol ester intermediate to the carboxyl terminus of ubiquitin. Using a combination of covalent affinity and high performance liquid chromatographic methods, the pututive E2 isozymes have been purified to apparent homogeneity. The E2(14kDa) isozyme resolved into two forms differing in net charge at pH 7.5. All of the E2 isozymes contained only one thiol ester site except for E2(17kDa) and E2(20kDa) which were capable of forming two such adducts per subunit. Thiol ester formation was rapid for the E2 isozymes and required the presence of activating enzyme. In contrast, the reverse reaction of thiol ester transfer from E2 to E1 was kinetically significant for only E2(14kDa), E2(20kDa), and E2(24kDa). The stability of E2(17kDa) and E2(32kDa) to such trapping may reflect a marked shift in binding affinity to E1 upon thiol ester formation. In addition, differential rates for thiol ester formation to each subunit of dimeric E2(14kDa) was also noted. The E2(14kDa) isoforms were approximately 10-fold more active in E3-dependent ubiquitin-protein ligation than either E2(20kDa) or E2(32kDa). Neither E2(17kDa) nor E2(24kDa) supported this reaction. In addition, the thiol ester formed to E2(14kDa) was inherently more reactive since its second order rate constant for the E3-independent transfer of ubiquitin to the small molecular weight nucleophile dithiothreitol was an order of magnitude greater than found for the other isozymes. If these proteins constitute a family of isozymes, they exhibit considerable catalytic diversity.