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浆结构域并介导其转运到溶酶体膜蛋白的胞浆因子，进一步阐明胞浆因子在溶酶体膜蛋白分选途径中的分子机制。将大鼠肝脏胞浆部分应用于GST和溶酶体膜蛋白胞浆结构域结合的融合蛋白LGP85柱上，用0.5M氯化钠洗脱胞液因子。通过SDS-PAGE分析，确定了相对分子质量为90,000和53,000的蛋白质。在这一纯化步骤中，使用辣根过氧化物酶标记的针对LGP85胞浆结构域的合成肽(HRP-LGP85C-Tail)，通过斑点印迹法检测与LGP85胞质结构域结合的胞浆因子的存在。通过分析CF90和CF53的N端序列，发现CF90和CF53的N端序列分别由16个氨基酸残基和11个氨基酸残基组成。此外，还发现CF90的N端序列与热休克蛋白90(HSP90)的N端序列完全相同，而CF53的N端序列与β-微管蛋白高度同源。利用针对HSP90或β-微管蛋白的特异性抗体的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作用于LGP85溶酶体的机制。

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.