Intracellular protein degradation carries out important cellular functions, such as the modulation of the levels of regulatory proteins and the removal of abnormal proteins. The long-term objective of this research is the elucidation of the biochemical mechanisms of protein breakdown. We have shown that a major system for selective protein breakdown is mediated by the polypeptide ubiquitin (Ub), which becomes covalently linked to proteins destined for degradation. We have delineated several enzymatic reactions in the formation and breakdown of Ub-protein conjugates and studied mechanisms by which certain proteins are recognized by the Ub ligation system. However, many important questions remained open concerning the mechanisms,selectivity and regulation of the Ub pathway. It is not clear what signals target most proteins for degradation. The mode of action of the protease complex that degrades proteins ligated to Ub is totally obscure, as is its integration with the action of isopeptidases that release Ub for reutilization. We shall study these problems with enzymes of a cell-free system from reticulocytes. In addition, we shall study the biochemical mechanisms of the regulation of the breakdown of cyclins, proteins that are rapidly degraded at specific stages of the cell cycle. Recent results indicate that cyclin degradation is carried out by the Ub system. With the use of a cell-free system from fertilized clam oocytes, we shall try to identify the enzymes involved in the ligation of Ub to cyclin and to purify the cyclin-specific Ub-protein ligase. Possible mechanisms of regulation, such as the activation of this ligase at the appropriate time of the cell cycle, or the conversion of cyclin to a form susceptible to the action of the ligase, will be examined. In view of its important cellular functions, the elucidation of the mode of action of the ubiquitin pathway is of basic importance and may help in the future understanding of diseases caused by abnormal protein degradation.
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