A COMPARISON OF UBIQUITIN-DEPENDENT PROTEOLYSIS OF ROD OUTER SEGMENT PROTEINS IN RETICULOCYTE LYSATE AND A RETINAL-PIGMENT EPITHELIAL-CELL LINE

We compared ATP- and ubiquitin-dependent proteolysis in supernatants o f rabbit reticulocyte lysate and a human retinal pigment epithelial (R PE) cell line. At pH 7.8, both preparations catalyzed the conjugation of [I-125]ubiquitin to endogenous proteins, generating an equivalent a mount of high mass (>150 kDa) [I-125]ubiquitin-protein adducts. Both p reparations degraded exogenous histone 2A, oxRNase and beta-lactoglobu lin in an ATP-dependent manner. Addition of ubiquitin (12 or 120 mu M) to reticulocyte lysate stimulated (1.4-fold) ATP-dependent degradatio n only of histone 2A. Addition of 12 mu M ubiquitin to RPE supernatant resulted in greater than or equal to 3-fold enhancement in degradatio n of all three substrates. Next, we compared the ability of the two pr oteolysis systems to degrade bovine rod outer segment (ROS) nonintegra l membrane proteins. [I-125]ROS protein degradation by reticulocyte ly sate was almost exclusively ATP-dependent and was completely inhibited by hemin and vanadate, inhibitors of ATP- and ubiquitin-dependent pro teolysis. RPE supernatant also degraded ROS proteins by an ATP-depende nt mechanism, and, unlike results obtained in reticulocyte assays, thi s degradation increased (2-fold) upon ubiquitin supplementation. Both proteolysis systems degraded ROS proteins of molecular mass approximat e to 10, 30, 37, 40 and 60 kDa, with coincident formation of high mass species. Reticulocyte lysate also degraded 100 and 150 kDa ROS protei ns, whereas RPE supernatant did not. The 10, 37 and 40 kDa species wer e identified by western blot as the gamma-, beta- and alpha- subunits of rod transducin (G(t)), respectively. RPE supernatant generated (som e) ROS proteolysis products that remained acid-precipitable. As compar ed with patterns of proteolysis in reticulocytes, RPE supernatant (1) degraded 100% more G(t beta gamma) (2) generated >10-fold the amount o f high mass (putative ubiquitin-ROS protein) conjugates and (3) prefer entially degraded G(t beta gamma) relative to G(t alpha). The ubiquiti n-dependent enhancement of ATP-dependent degradation of all proteins t ested in RPE supernatant makes the RPE system a valuable experimental tool for the explicit demonstration of ubiquitin-dependent proteolysis .