Evidence for ATP and ubiquitin dependent degradation of proteins in cultured bovine lens epithelial cells.
Degradation of endogenous lens proteins has been difficult to show under physiological conditions using lens tissue preparations. In contrast, active proteolytic systems in cultured bovine lens epithelial (BLE) cells have been demonstrated previously. BLE cells also contain ubiquitin, a 76 amino-acid polypeptide which is conjugated to proteins in an ATP/Mg(2+)-dependent process prior to their cytosolic proteolysis. In this study, we show that histone H2A, alpha-crystallin, and actin are conjugated to ubiquitin, resulting in higher molecular mass species, which are detected by anti-ubiquitin antibodies. These proteins are also degraded in cell-free assays containing BLE cell supernatants under physiological conditions in an ATP/Mg(2+)-dependent manner. Observation of 125I-labeled proteolytic fragments was made after SDS polyacrylamide gel electrophoresis of the assays. Quantitation of trichloroacetic acid-soluble radiolabeled fragments generated in the presence of ATP/Mg2+ revealed that, with BLE cell supernatant, 25% of the histone H2A was degraded in 3 hr. Proteolysis of alpha-crystallin and actin amounted to 2.3% and 2.9%, respectively. The requirement of ATP/Mg2+ for proteolysis and the observation of ubiquitin conjugation to the same proteins is consistent with the presence of a ubiquitin-dependent proteolytic pathway in BLE cells. Additionally, in this study the BLE cell proteases were even more active on some substrates than the reticulocyte ubiquitin/ATP-dependent proteolytic system.
Jahngen-Hodge
J
J
Laboratory for Nutrition and Cataract Research, USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111.
Laxman
E
E
Zuliani
A
A
Taylor
A
A
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002478:Cells, Cultured; D003459:Crystallins / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007903:Lens Capsule, Crystalline / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D014452:Ubiquitins / Q000378:metabolism*
D000199:Actins; D003459:Crystallins; D006657:Histones; D014452:Ubiquitins; D000255:Adenosine Triphosphate