Characterization of a novel keratinocyte ubiquitin carrier protein.
A novel member of the ubiquitin carrier protein family, designated E2EPF, 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 E2EPF 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, E2EPF 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 E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF 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 E2EPF involving auto- and multiubiquitination.
Liu
Z
Z
Department of Dermatology, Medical College of Wisconsin, Milwaukee 53226, USA.
Haas
A L
AL
Diaz
L A
LA
Conrad
C A
CA
Gíudice
G J
GJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D006868:Hydrolysis; D015603:Keratinocytes / Q000378:metabolism*; D008025:Ligases; D008239:Lysine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D002352:Carrier Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D008239:Lysine