ATP Serves Two Distinct Roles in Protein Degradation in Reticulocytes, and One Independent of Ubiquitin One Requiring

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 ~-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 12Sl-bovine serum albumin (BSA) were completely (9399%) 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 (twoto 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 AlP-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 heroin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway. Protein degradation within mammalian and bacterial cells requires metabolic energy (1, 2). The finding that ATP can stimulate proteolysis in cell-free extracts (2-4) has made possible appreciable progress in elucidating the basis for this energy requirement. In rabbit reticulocyte extracts, Etlinger and Goldberg (3) described a soluble, alkaline proteolytic system that is dependent on ATP. This nonlysosomal system appears responsible for the selective degradation of abnormal proteins as well as for the elimination of many normal proteins during reticulocyte maturation (5, 6). Hershko, Rose, and coworkers (7-10) have presented extensive evidence that multiple protein components are necessary for the stimulatory effect of ATP. One of these is ubiquitin (10), an 8,500-dalton polypeptide (11), which in the presence of ATP can be ligated to eamino groups of lysine residues of various cellular proteins (12). In this process, ATP-Mg ÷+ is required for the activation of the carboxyl glycine residue of ubiquitin to a form which can be linked by an isopeptide bond to lysines in proteins (13). In support of this model, Chin et al. (14) showed that the degree of attachment of ubiquitin to denatured hemoglobins correlated with their rates of degradation when these proteins were microinjected into HeLa ceils. Hershko et al. (2, 9) have argued that the formation of ubiquitin-protein conjugates is the explanation of the ATP requirement for protein breakdown. Thus ATP would be required not for protein hydrolysis but for an initial recognition reaction, in which the substrates undergo a modification that would enhance their susceptibility to degradation by cytosolic ATP-independent proteases. One reason that this novel explanation of the ATP effect was attractive was that no precedent existed for proteolytic enzymes whose function required ATP. However, in the last two years, ATP-dependent proteases have been isolated from Escherichia coli (15-17) and mammalian mitochondria (19), and have been shown to be responsible for the energy-dependent degradation of abnormal proteins both in intact bacteria (15, 19-21) and in mitochondria (22). These novel enzymes, protease La from E. THE JOURNAL OF CELL BIOLOGY • VOLUME 96 IUNE 1983 1580-1585 1 580 © The Rockefeller University Press. 0021-9525/83/06/1580/06 $1.00 on April 30, 2019 jcb.rupress.org Downloaded from http://doi.org/10.1083/jcb.96.6.1580 Published Online: 1 June, 1983 | Supp Info: