ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation.

Abstract

The heat-stable polypeptide (APF-1) required for ATP-dependent proteolysis in reticulocytes enters into high molecular weight conjugates upon incubation with the fraction of reticulocytes that is retained by DEAE-cellulose. Conjugate formation requires ATP and Mg2+ and is inhibited by N-ethylmaleimide. UTP and GTP are inactive. These properties are identical to those of ATP-dependent protein breakdown in the same system, suggesting that the conjugates are intermediates in this process. The APF-1 conjugates are stable in sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Sephadex C-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.Although most cellular proteins turn over rapidly, the enzymic reactions of protein degradation have not yet been identified. A basic feature of intracellular protein breakdown is its absolute requirement for cellular energy. Inhibitors of ATP production inhibit the degradation of liver proteins (1, 2), of tyrosine aminotransferase in hepatoma cells (3), of normal proteins in bacteria (4, 5) and cultured cells (6, 7), and of abnormal proteins in Escherichia coli (8, 9) and reticulocytes (10, 11). Recently several reports have shown an ATP requirement for protein breakdown in cell-free systems. ATP stimulates the degradation of abnormal proteins in crude soluble extracts of reticulocytes (10, 11) and E. coli (12), and a requirement for ATP has been found in the cleavage of bacteriophage X repressor in vitro (13,14).We have shown that the ATP-dependent proteolytic system from rabbit reticulocytes is composed of several required components. A heat-stable polypeptide of a relatively small size, (Mr -8000) designated ATP-dependent proteolysis factor 1 (APF-1), has been resolved (15). APF-1 has no proteolytic activity itself but stimulates ATP-dependent protein breakdown by a crude protein fraction eluted from DEAE-cellulose, fraction 11 (15). Fraction II has been resolved into two subfractions, both required for protein breakdown by the ATP-APF-1 system (16).To gain a better insight into the roles of the different factors and of ATP in protein breakdown, we have now purified APF-1, radiolabeled it, and observed its association with other reticulocyte components. ATP is required for binding of APF-1 to reticulocyte proteins and the binding seems to be covalent in nature. METHODS Preparation of Enzyme Fractions and Purification of APF-1. Lysates from ATP-depleted rabbit reticulocytes were prepared and separated on DEAE-cellulose into fraction I (unadsorbed material) and fraction 11 (0.5 M KCI eluate), as described earlier (15, 16). Fraction I was subjected to heat treatment (90'C, 20 min) and gel filtration on a column (1.5 X 90 cm) of Sephadex G-75, as described (15). Then 10 mg of this material was adsorbed onto a column (1.5 X 30 cm) of CM-Sephadex equilibrated with 10 mM potassium phosphate and was eluted with a 0-150 mM linear gradient of KCl. The active peak, designated APF-1, eluted at about 6...

Formation of conjugates by 125I‐labelled ubiquitin microinjected into cultured hepatoma cells

“…It is more difficult to interpret the significance of the bands found predominantly in the cytoplasm. Conjugates of ubiquitin with cellular proteins have been formed in extracts of reticulocytes [6,7], by the ATP-dependent linkage of the carboxyl terminal of ubiquitin to the lysine residues of other proteins via an isopeptide bond [7,16]. These conjugates were rapidly broken down in the absence of ATP [7].…”

“…It is widely distributed in animals, plants, yeasts and bacteria [1,2]. In animal cells ubiquitin has been found both in free form [1,4] or covalently bound by its carboxyl terminal to other proteins [5][6][7][8]. In the chromatin of interphase cells, ~10% of histone 2A and -,-1% of histone 2B are covalently linked to ubiquitin [8].…”

“…Amounts of ubiquitin linked to H2A decrease in nucleoli undergoing hypertrophy [ I 1 ], in 'active' chromatin [ 12] and during chicken erythrocyte maturation [ 13]. Ubiquitin may be involved in intracellular protein degradation in reticulocytes [7,[14][15][16]. Conjugates of ubiquitin with other proteins could act as substrates of intracellular proteolytic systems [6,7].…”

“…Ubiquitin may be involved in intracellular protein degradation in reticulocytes [7,[14][15][16]. Conjugates of ubiquitin with other proteins could act as substrates of intracellular proteolytic systems [6,7].…”

“…This was done essentially as in [6]. To 500/ag pure ubiquitin dissolved in 20/A 0.5 M phosphate buffer (pH 7.5), were added I0/A of a solution containing I mCi carrier-free Na12SI.…”