Membrane Recognition and Fusion Involved in Lysosome Formation

参与溶酶体形成的膜识别和融合

基本信息

批准号：
01480493
负责人：
KATO Keitaro
金额：
$ 4.42万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480493/
关键词：
Membrane traffic cDNA cloning Target signal Endosome Autolysosome Fusion エンドゾ-ム Mー6ーP受容体 M-6-P受容1本

项目摘要

Rat liver lysosomal membrane glycoprotein having a molecular weight of 107K (LGP107) was purified and antibodies against the protein were raised on rabbits. A conjugate of its Fab' fragment with horseradish peroxidase (HPR-antiLGP107Fab') was prepared as a probe for the subcellular antigen. Electronimmunocytochemistry in primary cultured rat hepatocytes showed that LGP107 resided primarily within lysosomes and was associated with luminal amorphous materials as well as limiting membranes. In addition, LGP107 was shown to be substantially distributed throughout the endocyctic vacuolar system. The glycoprotein was found clustered in coated pits at the cell surface and localized along the surrounding membranes in endocyctic vesicles. When cultured cells were exposed to HRP-antiLGP107 Fab', the antibody which was bound to its antigen within the coated pits was internalized via a system of endocyctic vesicles, and transported to lysosomes. These data suggest that LGP107 circulates between th … More e cell surface and lysosomes through the endocytic membrane traffic in hepatocytes.Four cDNA clones for lysosomal membrane glycoproteins such as acid phosphatase, LGP107, LGP96, and LGP85, were isolated to examine target signal of lysosomal membranes to lysosomes. Three of which except LGP85 have highly hydrophobic domain near their C terminals as a membrane anchoring domain and short cytosolic domain which may play a role to get information from outside of lysosomes. Also the anchoring domains including the short cytosolic tails of these three lysosomal membrane glycoproteins have a high sequence similarity (more than 60%) among them. However, LGP87 has two hydrophobic domains, suggesting two membrane anchoring domains at N and C terminus and a short cytosolic tail at C terminal. The membrane anchoring domain at N terminal is a signal peptide which is not cleaved off. Lysosomal membrane glycoproteins so far cloned showed no example such as LGP85 having two membrane anchoring domains. Fusions between purified endosomes and autolysosomes isolated from rat livers received either [^<125>I] asialofetuin or asialofetuin conjugated with HRP were examined and fusion products were determined as radioactive DAB polymers on milipore filters. The fusion between endosomes and autolysosomes showed that although endosome-endosome fusion requires ATP, endosome-autolysosome fusion proceeded without ATP. Less

将大鼠肝溶酶体膜糖蛋白纯化为107K（LGP107），并在兔子上饲养针对蛋白质的抗体。将其fab的“与辣根过氧化物酶（HPR-ANTILGP107FAB”）进行的结合物作为亚细胞抗原的探针。一级培养的大鼠肝细胞中的电子免疫细胞化学表明，LGP107在溶酶体内居住，与腔内无定形材料以及限制机制有关。此外，LGP107被证明在整个内吞液压系统中基本分布。发现将糖蛋白聚集在细胞表面的涂层坑中，并沿周围蔬菜的周围机理定位。当将培养的细胞暴露于HRP-抗Antilgp107 Fab'时，通过这些数据的系统将其与涂层凹坑内的抗体结合的抗体表明，LGP107在乙二醇中的内吞膜中循环的lgp107在……更多的E细胞表面和溶酶体之间循环。分离磷酸酶，LGP107，LGP96和LGP85，以检查溶酶体膜对溶酶体的靶信号。除LGP85外，其中三个在其C端子附近具有高度疏水结构域作为膜锚定结构域和短胞质结构域，这可能会起作用，从而从溶酶体外部获取信息。另外，锚定结构域包括这三种溶酶体膜糖蛋白的短胞质尾巴具有很高的序列相似性（超过60％）。然而，LGP87具有两个疏水结构域，表明在N和C末端，两个膜锚定结构域以及C末端的短胞质尾巴。 N端子处的膜锚定结构域是一种信号肽，未切断。到目前为止，克隆的溶酶体膜糖蛋白没有显示两个膜锚定域的LGP85。检查了从大鼠肝脏中分离出的纯化内体和自溶液中的自生体体之间的融合，检查了[^<125> i] asialofetuin或与HRP结合的ASIALOFETUIN，并检查了融合产物，并确定融合产物作为miripore滤波器上的放射性DAB聚合物。内体和自生体体之间的融合表明，尽管内体 - 粘体融合需要ATP，但内体 - 小气溶液体融合在没有ATP的情况下进行。较少的