MOLECULAR SORTING DURING INTRACELLULAR TRANSPORT

细胞内运输过程中的分子分选

• 批准号: 2175618
• 负责人: IRA S MELLMAN
• 金额: $ 25.01万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175618
• 关键词: MOLECULAR; SORTING; DURING; INTRACELLULAR; TRANSPORT

The plasma membranes of many mammalian cells are differentiated into biochemically and functionally distinct. This asymmetric arrangement of membrane components is best understood in the case of epithelial cells, whose surfaces are polarized into apical and basolateral domains that allow epithelia to serve as a selective barrier that regulates the interaction of tissues with the environment. Polarity in epithelial cells is generated by a series of molecular sorting events on the secretory and endocytic pathways that ensure the specific transport of newly synthesized, as well as internalized, membrane components to the appropriate plasma membrane domain. Despite the fundamental importance of these events, relatively little is known about the mechanisms by which endosomes and the Golgi complex distinguish between components destined for the apical and baolateral surfaces. During the previous grant period, we found that the sorting of membrane proteins in kidney epithelia, and probably other polarized cell types, is controlled by a novel set of distinct but generally distributed cytoplasmic domain determinants that specify transport to the basolateral plasma membrane. These determinants are often dependent on critical tyrosine or dileucine-containing motifs and thus bear some relationship with signals that specify localization at clathrin-coated pits. We also found that basolateral targeting determinants are dominant to, and hierarchically arranged with, a second generally distributed signal that specifies apical targeting: inactivation of the basolateral determinant invariably results in transport to the apical plasma membrane. In addition, the same basolateral signals are active in both the endocytic and secretory pathways, suggesting that similar or identical sorting strategies are used in both endosomes and the Golgi complex. Consequently, it seems likely that the sorting mechanisms used by epithelial cells may be widely conserved in different polarized cell types and throughout evolution. We plan to make use of this information and extend our work to elucidate the mechanism of polarized sorting at the cellular, biochemical, and molecular levels. Our Specific Aims include: (1) Characterize the structure, function, and distribution of basolateral sorting determinants; (2) Identify factors that regulate polarized sorting in intact cells; (3) Biochemically dissect the mechanism of sorting in endosomes through the use of cell-free assays that reconstitute receptor sorting and transport vesicle formation in vitro; (4) Identify and characterize cytosolic and/or coat proteins likely to be involved in endosome (or Golgi) function; (5) Characterize mammalian homologs of genes required for polarity during budding in yeast.

许多哺乳动物细胞的质膜分化为 生化和功能上不同。这种不对称的安排 在上皮细胞的情况下，最好理解膜成分， 其表面两极化成顶端和基底外侧域 允许上皮用作调节的选择性障碍 组织与环境的相互作用。上皮细胞的极性 由分泌性的一系列分子分类事件产生 确保新运输的内吞途径 合成的以及内部化的膜成分 适当的质膜结构域。尽管至关重要 这些事件，关于该机制的知之甚少 内体和高尔基体综合体区分注定的组成部分 对于顶端和鲍尔外侧表面。 在上一个赠款期间，我们发现膜的排序 肾上皮中的蛋白质以及可能其他极化细胞类型的蛋白质是 由一组新颖但普遍分布的新颖的人控制 胞质结构域的决定因素，该决定因素指定运输到基底外侧 质膜。这些决定因素通常取决于关键 酪氨酸或含二李酮的基序，因此具有一定的关系 带有指定在网格蛋白涂层坑中定位的信号。我们也是 发现基底外侧靶向决定因素是主导的，并且 与层次结构一起排列，第二个通常分布的信号， 指定顶点靶向：基底外侧决定因素的灭活 总是会导致运输到顶端质膜。在 此外，相同的基底外侧信号在两个内吞作用中均处于活动状态 和分泌途径，表明相似或相同的排序 策略都用于内体和高尔基体综合体中。最后， 上皮细胞使用的分选机制似乎可能 在不同的极化细胞类型和整个范围内都广泛保守 进化。我们计划利用这些信息，并将我们的工作扩展到 阐明在生化的细胞上极化分选的机理 和分子水平。我们的具体目的包括：（1）表征 基底外侧分选决定簇的结构，功能和分布； （2）确定调节完整细胞中极化分选的因素； （3） 从生化剖析内体中分选的机制 使用无细胞的测定，以重建受体分类和传输 体外形成囊泡； （4）识别和表征胞质和/或 外套蛋白可能参与内体（或高尔基体）功能； （5） 表征了极性所需基因的哺乳动物同源物 在酵母中萌芽。