Abstract
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.
Highlights
Ronald Hough and Martin,Rechsteiner From the Department of Biology, University of Utah, Salt Lake City, Utah84112
ATP-dependent proteolysis, 1251-lysozymewas incu- degradation
Ubiquitin is clearly implicated in proteolysis, its Purification of covalently modified lysozyme from hemin-inhibited lysates containing ‘251-ubiquitinand 1311-lysozymeconfirmed that both molecules were present in the complexes
Summary
Formation and Purfication of High Molecular Weight U b i - quitin-lysozyme Conjugates-Preliminary to purifying conjugates, we repeated several experiments reported by Haas and. ATP-dependent proteases have not yet been purified from eukaryotes, but here too progress has been made since it is known that ubiquitin plays a keyrole in intracellular proteolysis [4,5] Evidence for this includes the demonstration that theintracellular concentrationof ubiquiunits of creatine kinaselml; Buffer A, 10 mM Tris-HC1,pH 7.0,1mM dit~othreitol2,0%glycerol (v/v); Buffer B, 10 mM %is-HC1, pH 7.0, 1mM dithiothreitol, 25 mM KCI, 10 mM NaCl, 1.1 mM MgCI,, 0.1 mM Na ethylenediaminetetraaceticacid, and 20% glycerol (v/v); NEM, ~-ethylmaleimideS; DS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCA; trichloroacetic acid; BSA, bovine serum albumin. As with the formation of ubiquitin conjugates, there was no obvious changeinthe molecular weight distribution of lysozyme conjugates during incubation. Because these high molecular weight lysozyme-containing complexes were formedonly inthepresence of ATP and complexes were prepared as described under "Experimental Procedures" andanalyzed aboveby electrophoresis on 13% SDS-acrylamide gels.
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