A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of factors and evidence for ATP-dependent complex formation.
It was found previously that proteins conjugated to ubiquitin are degraded by an ATP-dependent enzyme system, but the mode of action of this system was unknown. We have resolved from reticulocyte extracts three factors that are required for the ATP-dependent breakdown of 125I-lysozyme-ubiquitin conjugates. Two of the factors interact with ATP, as shown by their protection against heat inactivation by the nucleotide. When the three factors are incubated with 125I-lysozyme-ubiquitin conjugates and ATP, there is a lag of 4-6 min in the formation of acid-soluble products before the onset of rapid proteolysis. The lag can be abolished by incubation of the three factors with MgATP prior to the addition of the substrate. This "activation" process does not take place if any of the three factors is omitted from preincubation (and added subsequently) or when ATP is replaced by a nonhydrolyzable analog. Analysis of size distribution by glycerol density gradient centrifugation showed that following incubation of the three factors with MgATP, a high molecular mass (greater than 1000 kDa) activity is formed. That the high molecular weight form is a complex of the three factors is indicated by the finding that its formation is accompanied by a corresponding decrease in the levels of the free forms of all three factors. Complex formation seems to be similar to the activation process with regard to time course, requirements for ATP and Mg2+, partial effect of CTP, and lack of effect of nonhydrolyzable ATP analogs. It is suggested that one role of ATP in conjugate breakdown is the formation of an active multienzyme complex.
Ganoth
D
D
Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Leshinsky
E
E
Eytan
E
E
Hershko
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002845:Chromatography; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
D011506:Proteins; D014452:Ubiquitins; D012710:Serum Albumin, Bovine; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D009113:Muramidase; D010447:Peptide Hydrolases; D008274:Magnesium