Demonstration of a factor in fraction I of reticulocyte lysates necessary for the steady state accumulation of ubiquitin conjugates of des-75-76-ubiquitin.
Addition of des-75-76-ubiquitin (ubiquitin lacking its two C-terminal glycine residues) to reticulocyte lysates leads to the inhibition of proteolysis and the formation of conjugates between it and native ubiquitin, as demonstrated by the incorporation of both 125I-labeled des-75-76-ubiquitin and 125I-labeled ubiquitin into these conjugates. Conjugate formation is blocked by methylation of the amino groups of des-75-76-ubiquitin, consistent with the concept that the conjugates represent attachment of the ubiquitin alpha-carboxyl group to amino groups of des-75-76-ubiquitin. The lack of significant direct competition for conjugate formation by typical ubiquitinatable proteolysis substrates or by des-73-76-ubiquitin, together with differences in conjugate formation between des-73-76-ubiquitin and des-75-76-ubiquitin demonstrated earlier, indicates that the enzyme involved recognizes the ubiquitin sequence as a substrate for ubiquitination. Increasing concentrations of native ubiquitin first increase and then reduce the steady state level of conjugates of the des-75-76-protein, the inhibitory effects of high concentrations consistent with competition by native ubiquitin for conjugate formation. Upon fractionation of reticulocyte lysates, a factor essential to the net synthesis of conjugates of des-75-76-ubiquitin was demonstrated to be present in Fraction I and to behave as a protein of molecular weight 38,000. The role in this system of a factor from Fraction I other than ubiquitin indicates that a novel pathway is involved.
Bamezai
S
S
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Breslow
E
E
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008745:Methylation; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000378:metabolism*
D014452:Ubiquitins; C070976:ubiquitin, des-(75-76)-; D010447:Peptide Hydrolases