Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis.
To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.
D049069:ATP-Dependent Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism; D006360:Heat-Shock Proteins; D006427:Hemin / Q000494:pharmacology; D006609:High Mobility Group Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012697:Serine Endopeptidases; D014452:Ubiquitins / Q000378:metabolism*
D006360:Heat-Shock Proteins; D006609:High Mobility Group Proteins; D014452:Ubiquitins; D006427:Hemin; D000255:Adenosine Triphosphate; D009113:Muramidase; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases