CHARACTERIZATION OF AUTOPHAGIC VACUOLAR MEMBRANES

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Autophagy is a process by which intracellular organelles are sequestered and degraded within the lysosomal vacuolar system. It is a biologically important pathway for the degradation of endogenous proteins, not only in differentiation, metamorphosis and aging but also in normal protein turnover. The detailed events in the regulation and mechanisms of autophagy are still unknown and form the basis of my long range research goals. In this project, I am proposing to study in rat liver the events of autophagic vacuole (AV) formation and maturation to a residual body, a form of secondary lysosome. My focus will be on the AV membrane since its origin and fate are not presently known. Since the vacuolar membranes appear to be derived from pre-existing cytoplasmic membranes, I will prepare and characterize organelle-specific antibodies (eg, anti-endoplasmic reticulum, anti-Gogi, anti-plama membrane and anti-lysosome) and use them in an immunological analysis of the vacuolar membranes. Using glucagon to stimulate and insulin to inhibit autophagy (individually, in selected combinations and at different temperatures), experimental conditions will be established which yield predominantly nascent AV's (pre-lysosomes) or predominantly hydrolytic AV's. Using the organelle-specific antibodies, the source of the double membrane that envelopes regions of cytoplasm to form nascent AV will be identified and the fate of this membrane followed through maturation of the AV. Immunofluorescence and immunoelectron microscopy (eg, immunoperoxidase or Protein A-gold techniques) will be used, followed by AV isolation and immunological characterization of the limiting membrane. Selected artificial effectors of autophagy (eg, ethionine to inhibit and vinblastine to stimulate) will also be examined to determine the generality of the findings with glucagon and insulin. Finally, I will examine the interrelationships between the prelysosomal compartments of the endocytic (exogenous proteins) and autophagic (endogenous proteins) pathways by following the fate of an endocytosed protein during enhanced autophagy under conditions in which vacuole fusion with lysosomes is inhibited (ie, 16 C).