Characterization of a novel keratinocyte ubiquitin carrier protein

Abstract

A novel member of the ubiquitin carrier protein family, designated E2(EPF), has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2(EPF) catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2(EPF) was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2(EPF) was shown here to be independent of the lysine 48 multiubiquitin linkage, This functional characterization of E2(EPF) revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2(EPF) involving auto- and multiubiquitination.