Bovine lens epithelial cells have a ubiquitin-dependent proteolysis system.
Lens cells must remove obsolete or damaged proteins produced during development, maturation and aging to maintain lens transparency. In reticulocytes removal of abnormal or obsolete proteins is thought to involve a ubiquitin-dependent proteolytic pathway. Two hallmarks of ubiquitin (Ub) dependent proteolysis have previously been demonstrated in lens cell or tissue supernatants: (1) the presence of ubiquitin conjugates, and (2) ATP-dependent proteolysis. Nevertheless, conclusive proof was lacking of a requirement for ubiquitination of substrate proteins for proteolysis. Here we show that in bovine lens epithelial cell (BLEC) supernatant, ATP-dependent proteolysis is also ubiquitin-dependent. Ubiquitin-activating enzyme (E1), the first enzyme in the cascade of ubiquitin ligation, was purified over 3000-fold from a rabbit reticulocyte lysate using Ubiquitin-Sepharose, and showed ATP-PPi exchange activity. Antiserum to E1 was prepared in goats and affinity-purified on Protein G-Sepharose. Western blot analysis revealed that both the goat antiserum and purified antibody (anti-E1(IgG)) recognize specifically E1. Anti-E1(IgG) inhibits 86% of the ATP-dependent degradation of labeled histone H2A in reticulocyte lysate and 75% of the ATP-dependent degradation in BLEC. Upon reconstitution with purified E1, 100% and 80% of proteolysis was restored in reticulocytes and BLEC supernatant, respectively. This confirms that there is a ubiquitin-dependent proteolytic system in lens.
Laboratory for Nutrition and Vision Research, Tufts University, Boston, MA 02111.
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Huang
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Jahngen-Hodge
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Taylor
D000818:Animals; D000906:Antibodies; D002417:Cattle; D004848:Epithelium / Q000201:enzymology; D006657:Histones / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D008025:Ligases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology / Q000276:immunology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D000906:Antibodies; D006657:Histones; D011506:Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes