Studies of the Degradation of Proteins in Animal and Bacterial Cells

Summary

Proteins within animal and bacterial cells vary widely in their rates of degradation, and these differences appear to reflect differences in protein conformations. For example, half-lives of intracellular proteins as well as serum appear to correlated with their inherent sensitivity to various proteolytic enzymes. We have also found that the half-lives of proteins are related to their net charge. In various tissues and in rat serum, proteins with acidic isoelectric points are degraded more rapidly that neutral or basic ones. Isoelectric point and subunit molecular weight are two independent parameters that influence degradative rates of proteins.

Protein breakdown in animal and bacterial cells also helps to protect the cell against accumulation of abnormal polypeptides. E. coli for example selectively degrade incomplete proteins that result from nonsense mutations or proteins containing amino acid analogs. Similarly mammalian reticulocytes rapidly degrade abnormal globins that contain valine or lysine analogs. This degradative process in mammalian tissues generally resembles that in E. coli. For example in E. coli and reticulocytes the degradative process requires metabolic energy.

In an attempt to define the mechanisms of protein degradation, we have investigated the rapid degradation of X-90, a fragment of β —galactosidase resulting from a nonsense mutation. A smaller polypeptide that contains the original amino terminal sequence has been found that is an intermediate in X-90 degradation. When energy metabolism was inhibited, the loss of X-90 and of the smaller fragments was also inhibited. Thus energy is required not only for the initial cleavage but also for subsequent proteolytic steps. The initial cleavage in the degradative process seems to be due to an endoproteolytic cleavage. Deg T ^- mutants defected in degradation of abnormal proteins have a reduced capacity to carry out this reaction.

Proteins within animal and bacterial cells vary widely in their rates of degradation (1–3). These differences in protein half-lives must have important implications for the control of cell metabolism (2,3). For example, in rat liver those proteins with short half-lives tend to be rate-limiting enzymes in metabolic pathways (2). This rapid degradation insures that the intracellular levels of such enzymes can fluctuate rapidly in response to environmental changes (3). Thus protein half-lives appear to have evolved in part to regulate the flux of substrates through metabolic pathways (3). Such considerations also imply that the variations in the half-lives of different proteins are determined by differences in protein structure. In fact there is now appreciable experimental evidence (1,2) that rates of degradation must be encoded in the primary sequence of the polypeptide along with information for its catalytic and allosteric functions. However, the exact structural features of proteins that influence their degradative rates are still not clear.