title: ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.
abstract: Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to epsilon-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 125I-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two- to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP-stimulated proteolysis. The ATP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg++ and ATP hydrolysis and are inhibited by hemin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D006427:Hemin / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014452:Ubiquitins
chemical_list: D002364:Caseins; D002868:Chromosomal Proteins, Non-Histone; D011506:Proteins; D014452:Ubiquitins; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D006427:Hemin; D000255:Adenosine Triphosphate; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate