STUDIES ON THE MECHANISM OF AUTOPHAGY

自噬是细胞内细胞器被 在溶酶体空泡系统中隔离和降解。 它是生物上重要的降解途径。 内源蛋白质，不仅在分化、变态中 而衰老也在蛋白质的正常周转中。详细的事件 在自噬的调控和机制上仍不清楚 并构成了我长期研究目标的基础。在这 项目，我已经提议在大鼠肝脏中研究 自噬空泡(AV)的形成和成熟为次级 溶酶体。由于液泡的形成和随后的融合 由于溶酶体是膜事件，我的重点将放在 动静脉膜的表征。液泡膜 似乎是从先前存在的细胞质膜衍生而来。 使用细胞器特异性抗体与 免疫过氧化物酶细胞化学，我已经证明了这些 液泡不是由高尔基体或质膜形成的 成分，但它们很可能来自RER和 溶酶体。我现在要准备针对特定的单特异性抗体 膜蛋白的粗面内质网和溶酶体证明我最初 更详细地发现和研究自噬的途径。 应用高血糖素促进空泡形成和低血糖 温度抑制溶酶体水解酶的进入，我有 已建立的条件导致新的 形成的病毒。我现在使用单特异性抗体(即， 在电子显微镜水平上的免疫金定位)到 识别特定的膜抗原并确定是否所有的病毒都是 从RER形成的或者如果存在二次源(例如， 这些液泡的溶酶体)。我的结果表明 房室是由粗面内质网的光滑膜区形成的，即 也将确定是否在失去 膜结合多核糖体与自噬的开始。这就做 首先测量肝脏中膜结合核糖体的水平 在营养丰富的情况下灌流(抑制自噬)和 营养耗竭(自噬刺激)条件，然后 比较稳定条件下的自噬反应 (环己亚胺)或不稳定(组氨醇)多核糖体。最后， 我将研究细胞损伤对肝脏自噬的影响， 为了确定自噬反应是否类似于 这是生理刺激时的特征。对……的调查 可能促进这一反应的替代条件将是 变得重要，在评估 自噬的调节。

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.