Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.
It was previously shown that ubiquitin is very similar to the polypeptide cofactor of the ATP-dependent protein degradation system from rabbit reticulocytes (Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Biol. Chem. 255, 7529-7532). We have extended this work to show that the peptic peptide maps are identical for bovine ubiquitin and the polypeptide cofactor isolated from human erythrocytes. It was noted however that ubiquitin preparations were less active in stimulating proteolysis than preparations of the polypeptide cofactor. This decreased activity has been shown to be due to the presence of an inactive form of ubiquitin in some preparations. The two forms of ubiquitin are separable by high performance liquid chromatography. The active form of ubiquitin has the COOH-terminal sequence -Arg-Gly-Gly at residues number 74 to 76. The inactive form terminates in -Arg74 as previously reported in the sequence studies of ubiquitin. Limited tryptic digestion of active ubiquitin yields the inactive, later eluting form and the dipeptide glycylglycine. This preteolytic cleavage apparently occurs during purification from most tissues. We thus propose reserving the term ubiquitin for the intact 76-amino acid sequence and designating the 74-amino acid sequence as ubiquitin-t to indicate its derivation by a tryptic-like protease cleavage. This 76-residue sequence is consistent with the covalent structure of protein A-24, a conjugate where carboxyl group of the COOH-terminal glycylglycine of ubiquitin is linked by an amide bond to the epsilon-amino group of Lys-119 of histone H2A. Thus, the structural requirements of the protein and ubiquitin molecules are identical for formation of protein A-24 and for forming the covalent conjugates thought to be intermediates in ATP-dependent protein degradation.
D000255:Adenosine Triphosphate / Q000097:blood; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine; D002868:Chromosomal Proteins, Non-Histone / Q000097:blood*; D005998:Glycine; D007700:Kinetics; D009698:Nucleoproteins / Q000097:blood*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014452:Ubiquitins
D000596:Amino Acids; D002868:Chromosomal Proteins, Non-Histone; D009698:Nucleoproteins; D010446:Peptide Fragments; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D001120:Arginine; D005998:Glycine