ATTACHMENT OF RIBOSOMES TO MICROSOMAL MEMBRANES

本项目的长期目标是了解 负责亚细胞分布的生物发生机制 细胞膜和细胞器中的特定蛋白质。 我们将重点 在分子方面的机制，决定了 相对于膜磷脂双层的配置 在ER中合成的多肽。 我们还将研究排序 参与蛋白质在内质网中滞留的过程 或决定其转移到高尔基体，并从 到溶酶体或质膜。 细胞色素P-450， 和内质网的膜蛋白， 粘附地插入膜中，但大部分保留在 暴露在其细胞质表面，已被选为模型 蛋白质的一些工作。 基本结构特征 使P-450中未切割的插入信号停止 转移远端序列并将成熟蛋白锚在 磷脂双层，将通过检查 嵌合和改变的多肽的膜插入 在体外转录-翻译系统中产生 用基因工程模板编程 这些 研究还将提供深入了解其他 未切割的信号，其不具有halt传递功能， 要么穿过膜要么永久地 关于翻译后的研究 插入ER的蛋白质的分选，包括：1）分离 移行池的生物化学特征 为蛋白质从内质网输送到 高尔基体 在这些研究中， 在这种细胞器中积累的异常嵌合蛋白质， 导致其过度发展将被采用。 尝试将 用于识别载体的受体， 嵌合蛋白，可能积累在这些过度发育的 蓄水池 2)β-半乳糖苷酶结构特征的鉴定 负责甘露糖磷酸化的葡萄糖醛酸酶 这些残基决定了这种蛋白质在 溶酶体 3)一个解释的途径，其次是 溶酶体膜蛋白对溶酶体的作用及其鉴定 将这个蛋白质定位到它的目的地。 4)识别导致保留的特征 细胞色素P-450在内质网膜和微粒体 ER管腔内的酯酶。

The long term objective of this project is to understand the biogenetic mechanisms responsible for the subcellular distribution of specific proteins in membranes and organelles. We will focus on molecular aspects of the mechanisms that determine the disposition with respect to the phospholipid bilayer of membrane polypeptides synthesized in the ER. We will also study the sorting processes that are involved in the retention of proteins in the ER or determine their transfer to the Golgi apparatus, and from there, to lysosomes or the plasma membrane. Cytochrome P-450, and integral membrane protein of the ER which is cotranslationally inserted in the membrane but remains largely exposed on its cytoplasmic surface, has been chosen as a model protein for some of this work. The essential structural features that enable the uncleaved insertion signal in P-450 to halt transfer of distal sequences and to anchor the mature protein in the phospholipid bilayer, will be identified by examining the membrane insertion of chimeric and altered polypeptides generated in in vitro transcription-translation systems programmed with genetically engineered templates. These studies will also provide insights into the features of other uncleaved signals, which do not have halt transfer function but either pass through the membrane or remain permanently anchored to it. The studies concerning the posttranslational sorting of proteins inserted in the ER, include: 1) The isolation and biochemical characterization of the transitional cisternae that provide the site of exit for proteins delivered from the ER to the Golgi apparatus. for these studies, cells that synthesize an abnormal chimeric protein that accumulates in this organelle and leads to its overdevelopment will be employed. Attempts will be made to identify receptors of carriers that, complexed to the chimeric protein, may accumulate within these overdeveloped cisternae. 2) The identification of structural features in beta- glucuronidase responsible for the phosphorylation of the mannose residues that determine the sequestration of this protein in lysosomes. 3) an elucidation of the pathway followed by a lysosomal membrane protein to lysosomes and the identification of the sorting signal that address this protein to its destination. 4) An identification of the features that lead to the retention of cytochrome P-450 within the ER membrane and of a microsomal esterase within the ER lumen.