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

本研究的目的是鉴定识别溶酶体膜蛋白胞质区并介导转运至溶酶体的胞质因子，进一步阐明胞质因子在溶酶体膜蛋白分选途径中的分子机制。用0.5M NaCl洗脱胞质因子。经SDS-PAGE分析，鉴定出分子量为90，000和53，000的蛋白质。在该纯化步骤中，使用辣根过氧化物酶缀合的针对LGP 85的胞质结构域的合成肽（HRP-LGP 85 C-尾），通过斑点印迹法测定与LGP 85的胞质结构域结合的胞质因子的存在。发现胞质因子与HRP-LGP 85 C-尾的结合是特异性的，因为这种结合在过量的合成肽的存在下被抑制。当分析CF 90和CF 53的N-末端序列时，确定CF 90和CF 53的N-末端序列分别为16个氨基酸残基和11个氨基酸残基。此外，发现CF 90的N-末端序列与热休克蛋白90（HSP 90）的N-末端序列相同，CF 53的N-末端序列与β-微管蛋白的N-末端序列高度同源。HSP 90和β-tubulin参与LGP 85溶酶体靶向和功能的机制目前正在研究中。

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.