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

纯化分子量为107K的大鼠肝溶酶体膜糖蛋白(LGP107)，并在兔上产生针对该蛋白的抗体，制备其Fab'片段与辣根过氧化物酶的缀合物(HPR-抗LGP107Fab')作为亚细胞的探针。原代培养的大鼠肝细胞中的电免疫细胞化学显示，LGP107 主要存在于溶酶体中并且此外，LGP107 与管腔无定形材料以及限制膜相关，发现糖蛋白大量分布在细胞表面的包被凹坑中，并沿着内吞囊泡的周围膜定位。当培养的细胞暴露于 HRP-antiLGP107 Fab' 时，在包被的凹坑内与其抗原结合的抗体通过以下系统被内化：这些数据表明，LGP107 通过肝细胞中的内吞膜运输在细胞表面和溶酶体之间回收。溶酶体膜糖蛋白的四个 cDNA 克隆，例如酸性磷酸酶、LGP107、LGP96 和 LGP85，被分离以检查溶酶体膜的目标信号除 LGP85 外，其中三个在其 C 末端附近具有高度疏水性结构域作为膜锚定结构域和短胞质结构域，其可能发挥从溶酶体外部获取信息的作用。锚定结构域包括这三种溶酶体的短胞质尾部。膜糖蛋白之间具有很高的序列相似性（超过 60%），然而，LGP87 有两个疏水结构域，表明在 N 和 C 处有两个膜锚定结构域。末端和C末端的短胞质尾部是信号肽，迄今为止克隆的溶酶体膜糖蛋白没有显示出具有两个膜锚定结构域和纯化的内体之间的融合的例子。检查从接受[^ 125 I]脱唾液酸胎球蛋白或与HRP缀合的脱唾液酸胎球蛋白的大鼠肝脏分离的自溶酶体并融合内体和自溶酶体之间的融合表明，虽然内体-内体融合需要 ATP，但内体-自溶酶体融合在没有 ATP 的情况下进行较少。