The function of ubiquitin in intralysosomal protein degradation.

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HA-c-Cbl was expressed in HEK293 cells and immunoprecipitated. Insulin receptor expressed in HIRc cells was immunoprecipitaed. Then Both immunoprecipitants were mixed with E1,UbcH7 as an E2,ATP and its regenerating system, ubiquitin or its mutants. These mixtures were reacted in pH6.0, 6.5 and 7.2. Ubiquitinated insulin receptor were detected by immunoblot. When the reaction was done at pH7.2, insulin receptor were monoubiquitinated with multiple lysine residues in insulin receptor, whereas when the reaction was carried out at pH6.0, the insulin receptor was subjected to polyubiqutination through Lys-48 in ubiquitin molecules. Therefore, I projected next to analyze the structural change of recombinant c-Cbl between neutral pH and acidic pH.Next, I was planed to analyze the ubiquitination profiles of epidermal growth factor receptor and platelet derived growth factor receptor. These projects would be under going.Recently, I identified the monoubiquitination of LDH-A, a cytosolic protein, under oxidative stress was sequestered into and degraded in lysosomes. Now, I would search the ubiquitin receptor on the cytosolic surface of lysosomal membranes.