We compared ATP- and ubiquitin-dependent proteolysis in supernatants of rabbit reticulocyte lysate and a human retinal pigment epithelial (RPE) cell line. At pH 7.8, both preparations catalyzed the conjugation of [125I]ubiquitin to endogenous proteins, generating an equivalent amount of high mass (> 150 kDa) [125I]ubiquitin-protein adducts. Both preparations degraded exogenous histone 2A, oxRNase and beta-lactoglobulin in an ATP-dependent manner. Addition of ubiquitin (12 or 120 microM) to reticulocyte lysate stimulated (1.4-fold) ATP-dependent degradation only of histone 2A. Addition of 12 microM ubiquitin to RPE supernatant resulted in > or = 3-fold enhancement in degradation of all three substrates. Next, we compared the ability of the two proteolysis systems to degrade bovine rod outer segment (ROS) nonintegral membrane proteins. [125I]ROS protein degradation by reticulocyte lysate was almost exclusively ATP-dependent and was completely inhibited by hemin and vanadate, inhibitors of ATP- and ubiquitin-dependent proteolysis. RPE supernatant also degraded ROS proteins by an ATP-dependent mechanism, and, unlike results obtained in reticulocyte assays, this degradation increased (2-fold) upon ubiquitin supplementation. Both proteolysis systems degraded ROS proteins of molecular mass approximately 10, 30, 37, 40 and 60 kDa, with coincident formation of high mass species. Reticulocyte lysate also degraded 100 and 150 kDa ROS proteins, whereas RPE supernatant did not. The 10, 37 and 40 kDa species were identified by western blot as the gamma-, beta- and alpha- subunits of rod transducin (Gt), respectively. RPE supernatant generated (some) ROS proteolysis products that remained acid-precipitable. As compared with patterns of proteolysis in reticulocytes, RPE supernatant (1) degraded 100% more Gt beta gamma, (2) generated > 10-fold the amount of high mass (putative ubiquitin-ROS protein) conjugates and (3) preferentially degraded Gt beta gamma relative to G t alpha. The ubiquitin-dependent enhancement of ATP-dependent degradation of all proteins tested in RPE supernatant makes the RPE system a valuable experimental tool for the explicit demonstration of ubiquitin-dependent proteolysis.