CHARACTERIZATION OF AUTOPHAGIC VACUOLAR MEMBRANES

自噬是细胞内细胞器被隔离的过程 并在溶酶体空泡系统内降解。 从生物学上讲， 内源性蛋白质降解的重要途径，不仅在 分化，变态和衰老，而且在正常蛋白质 周转 详细的事件的调节和机制， 自噬仍然是未知的，并形成了我的长期研究的基础 目标. 在这个项目中，我建议在大鼠肝脏中研究 自噬泡（AV）的形成和成熟为残留体，一种形式 次级溶酶体 我的重点将放在AV膜上，因为它 起源和命运目前尚不清楚。 由于空泡膜 似乎是来自预先存在的细胞质膜，我会 制备和表征细胞器特异性抗体（例如， 抗内质网、抗Gogi、抗质膜和 抗溶酶体），并将其用于空泡的免疫分析中。 膜。 用胰高血糖素刺激和胰岛素抑制自噬 （单独地，以选定的组合和在不同的温度下）， 将建立实验条件， 新生AV（前溶酶体）或主要水解AV。 使用 细胞器特异性抗体，双膜的来源， 将鉴定形成新生AV的细胞质包膜区域， 该膜的命运跟随AV的成熟。 免疫荧光和免疫电子显微镜（例如，免疫过氧化物酶或 将使用蛋白A-金技术），然后进行AV分离和 限制膜的免疫学表征。 选择 自噬的人工效应物（例如，抑制自噬的乙烯利和长春碱 刺激）也将被检查，以确定的一般性， 胰高血糖素和胰岛素的结果。 最后，我将研究 内吞蛋白的前溶酶体区室之间的相互关系 （外源蛋白）和自噬（内源蛋白）途径， 在增强的自噬过程中， 在液泡与溶酶体融合被抑制的条件下（即， 16 C）。

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).