Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

Abstract

The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway, (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins, (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2's) except E2 20K and E2 230K. Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted.