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 mechanism of autophagy are still unknown and form the basis of my long range research goals. In this project, I have proposed to study in rat liver the events of autophagic vacuole (AV) formation and maturation into secondary lysosomes. Since formation of the vacuoles and subsequent fusion with lysosomes are membrane events, my focus will be on the characterization of the AV membrane. The vacuolar membranes appear to be derived from pre-existing cytoplasmic membranes. Using organelle-specific antibodies combined with immunoperoxidase cytochemistry, I have shown that these vacuoles are not formed from Golgi or plasma membrane constituents but that they are probably derived from RER and lysosomes. I will now prepare monospecific antibodies to specific membrane proteins of the RER and lysosome to prove my initial finding and examine in more detail the pathway of autophagy. Using glucagon to promote vacuole formation and low temperature to inhibit entry of lysosomal hydrolases, I have established conditions which result in the accumulation of newly formed AV's. I will now use monospecific antibodies (i.e., immunogold localization at the electron microscope level) to identify specific membrane antigens and determine if all AV's are formed from the RER or if there is a secondary source (e.g., lysosome) of these vacuoles. With my results indicating that the AV's are formed from smooth membrane regions of the RER, I will also determine if there is a relationship between the loss of membrane-bound polyribosomes and the onset of autophgy. I will first measure the levels of membrane-bound ribosomes in livers perfused under nutrient-enriched (autophagy inhibited) and nutrient-depleted (autophagy stimulated) conditions and then compare the autophagic response under conditons that stabilize (cycloheximide) or destabilize (histidinol) polyribosomes. Finally, I will examine the effects of cell injury on autophagy in the liver, in an effort to determine if the autophagic response is similar to that characterized during physiological stimuli. A survey of alternative conditions which may promote this response will become important in an evaluation of the mechanisms for the regulation of autophagy.
