Nearly all aspects of cell growth and metabolism rely upon precise control of protein levels. This balance is not achieved solely through the regulation of protein synthesis, but depends as well upon the rates of protein degradation. Metabolic shifts, environmental stress, and cellular differentiation all are accompanied bby selective proteolysis as the cell adapts to its new condition. To accomplish this, the machinery for intracellular proteolysis must possess both specificity and great flexibility. In eukaryotes, one pathway of intracellular proteolysis involves a 76 amino acid polypeptide, ubiquitin (Ub), which is attached covalently to proteins prior to their degradation. Two questions concerning this system will be addressed: what features of the target protein make it a substrate for ubiquitination, and what features of the resultant conjugate are recognized for subsequent proteolysis. A series of native and modified forms of cytochrome c's and bovine pancreatic trypsin inhibitor will be used as substrates to examine: 1) selectivity among a well-defined set of target proteins that represent folded, misfolded and unfolded conformations; 2) characteristics of ubiquitination sites -- sequence, tertiary structure, and flexibility; 3) involvement of the substrate N-terminus in target recognition; 4) the apparent cooperativity or processivity of ubiquitination. The structures of free Ub and various Ub-amide and Ub-protein conjugates will be compared by one- and two-dimensional 1H nuclear magnetic resonance spectroscopy. Ubiquitinated degradation intermediates of apo-cytochrome c will be sought in yeast. Enzymes of the yeast Ub pathway will be purified and an in vitro ubiquitination system established. Ub conjugation to various mutant apo-cytochrome c's in vitro will be compared with their in vivo turnover rates.
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