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IDENTIFICATION AND ROLE OF CYTOSOLIC FACTORS THAT PARTICIPATE IN TRANSLOCATION OF LYSOSOMAL MEMBRANE PROTEINS

参与溶酶体膜蛋白易位的胞质因子的鉴定和作用

基本信息

批准号：
07680770
负责人：
TANAKA Yoshitaka
金额：
$ 1.41万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680770/
关键词：
LYSOSOME ENDOSOME SORTING SIGNAL ソーティング・シグナル

项目摘要

The aim of this study is to identify cytosolic factors that recognize the cytoplasmic domain of lysosomal membrane proteins and mediate transport to the lysosomes, and further to elucidate molecular mechanisms of cytosolic factors in sorting pathway of lysosomal membrane proteins.When the cytosol fraction prepared from rat livers was applied to the column conjugated fusion proteins of GST and cytoplasmic domain of lysosomal membrane protein, LGP85, cytosolic factors were eluted with 0.5M NaCl. From analysis by SDS-PAGE,proteins having molcularweight of 90,000 and 53,000 were identified. In this purification step, the presence of cytosolic factors that bind to cytoplasmic domain of LGP85 was assayd by dot blotting using horseradish peroxidase-conjugated synthetic peptides against cytoplasmic domain of LGP85 (HRP-LGP85C-tail). It was found that binding of cytosolic factors and HRP-LGP85C-tail is specific, because this binding was inhibited in the presence of excess amounts of synthetic peptides.When analyzed N-terminal sequences of both CF90 and CF53, CF90 and CF53 were determined N-terminal sequences of 16 amino acid residues and 11 amino acid residues, respectively. Furthermore, it was found that N-terminal sequences of CF90 were identical with those of heat shock protein 90 (HSP90), and N-terminal sequences of CF53 represented highly homology to those of beta-tubulin. This was further confirmed by immunoblotting analysis using specific antibodies against HSP90 or beta-tubulin.Mechanisms by which HSP90 and beta-tubulin participate in lysosomal targeting and function in lysosomes of LGP85 are examining at present.

本研究旨在鉴定识别溶酶体膜蛋白胞质结构域并介导转运至溶酶体的胞质因子，进一步阐明溶酶体膜蛋白分选途径中胞质因子的分子机制。将大鼠肝脏制备的细胞质组分应用于GST和溶酶体膜蛋白细胞质结构域LGP85的柱共轭融合蛋白，用0.5M NaCl洗脱细胞质因子。通过SDS-PAGE分析，鉴定出分子量为90,000和53,000的蛋白质。在这一纯化步骤中，利用辣根过氧化物酶偶联的抗LGP85胞质结构域合成肽（HRP-LGP85C-tail），通过点印迹法检测结合LGP85胞质结构域的胞质因子的存在。研究发现，胞质因子与HRP-LGP85C-tail的结合是特异性的，因为这种结合在过量合成肽的存在下被抑制。在分析CF90和CF53的n端序列时，分别确定了CF90和CF53的16个氨基酸残基和11个氨基酸残基的n端序列。此外，CF90的n端序列与热休克蛋白90 （HSP90）的n端序列相同，CF53的n端序列与β -微管蛋白的n端序列高度同源。使用抗HSP90或β -微管蛋白的特异性抗体进行免疫印迹分析进一步证实了这一点。目前正在研究HSP90和β -微管蛋白参与溶酶体靶向和溶酶体功能的机制。