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

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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.