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

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 its 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 G-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.