MICROINJECTION STUDIES ON PROTEIN DEGRADATION

本建议的长期目标是通过以下方式阐明这些机制

The long term objective of this proposal os to elucidate the mechanisms by which cells selectively degrade their intracellular proteins and to discover the features of protein that promote their catabolism. Two cell systems are available in which it is possible to eliminate either proline endopeptidase or ubiquitin (Ub) conjugation. Individual, radiolabeled proteins will be injected into each cell line to determine which proteins are substrates for these pathways. This procedure has already revealed that ubiquitin and bovine serum albumin ar stabilized in cells lacking proline endopeptidase and that oxidized hemoglobin is stabilized in ts85 mouse cells which contain a labile Ub-activating enzyme. The half-lives of a series of temperature-sensitive T4 lysozymes will also be measured to test the hypothesis that thermodynamic stability correlates with metabolic stability. Surprisingly, T4 lysozyme is degraded very rapidly in HeLa cells, and we will determine whether surface cysteines or arginine-arginine pairs are responsible for its short life. In addition, specific proteins will be targeted to the nucleus to discover whether their degradation rates are affected by mislocalization. Finally, ubiquitin will be injected into ts85 mouse cells or HeLa cells and several aspects of Ub metabolism will be measured, including Ub's role in autophagy and release of injected proteins, as well as the subcellular location of Ub conjugates before and after heat-shock.