MEMBRANE INTERACTIONS IN EUKARYOTIC CELLS

我们建议研究生物发生关系和功能相互作用 这些细胞内隔室的膜共同构成 分泌途径(或内质网-质膜途径)。 这些隔室包括内质网、一系列高尔基体 隔室、分泌空泡(或颗粒)和不同载体 囊泡沿着这条通路在不同的连接处活跃，从而导致 质膜上的放电部位。 每当有需要时，我们建议通过以下方式改进细胞分级程序 使用特定的配体(例如抗体或凝集素)--而不是一般的 物理特性--用于亚细胞成分的分离。这个 获得的细胞组分将用于分离和纯化其 膜。这些膜的蛋白质将被分析和鉴定。 通过适当的程序，如凝胶电泳法，免疫覆盖法， 免疫沉淀，其他生物化学特定的相互作用，可以 通过凝胶或凝胶转移进行。选定的单元格类型 系统的研究是小鼠的红细胞(正常或 转化)和大鼠肝细胞。原生生物的膜蛋白 感兴趣的是：1)小鼠血糖素(用于红细胞)和2) 分泌成分(用于肝细胞)。其他膜蛋白将是 在时间上选择。在每种情况下，这些蛋白质遵循的途径 从它们的合成场所到它们的最终功能居住地 (或排放)将被遵循，每一次 移位后修饰将被确定。有了这个信息 安全，将尝试分析涉及到的机制 控制细胞内的囊泡交通--同时-- 在控制相互作用的膜之间的化学专一性方面。

We propose to study biogenetic relations and functional interactions among the membranes of those intracellular compartments that together constitute the secretory pathway (or the endoplasmic reticulum-plasmalemma pathway). These compartments include the endoplasmic reticulum, a series of Golgi compartments, secretory vacuoles (or granules) and different carrier vesicles active at different junctions along this pathway which leads to discharge sites on the plasmalemma. Whenever needed, we propose to improve cell fractionation procedures by using specific ligands (e.g. antibodies or lectins) - rather than general physical properties - for the isolation of subcellular components. The cell fractions obtained will be used to isolate and purify their membranes. The proteins of these membranes will be analyzed and identified by appropriate procedures e.g., gel electrophoresis, immuneoverlays, immunoprecipitation, other biochemically specific interactions, that can be carried out on gels or gel transfers. The cell types selected for systematic investigation are the murine erythroblasts (normal or transformed) and the rat hepatocyte. The membrane proteins of primary interest are: 1) the murine glycophorins (for erythroblasts) and 2) the secretory component (for hepatocyte). Other membrane proteins will be selected in time. In each case, the pathway followed by these proteins from their sites of synthesis to their sites of final functional residence (or discharge) will be followed and the compartments involved in each postranslocational modification will be identified. With this information secured, attempts will be made to analyze the mechanisms involved in the control of intracellular vesicular traffic and - in conjunction with it - in the control of chemical specificity among interacting membranes.