| 摘要 |
A method is provided for identifying an compound that affects an activity of a polypeptide subunit of a SCF complex. The method includes contacting a sample comprising a chimeric SCF complex assembled from subunits derived from Saccharomyces cerevisiae or human and another species and a CDC34p polypeptide with the compound under conditions that allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a SCF substrate. The ubiquitination of the SCF substrate is measured. A chimeric in vitro assay system is provided for measuring CDC53p or CUL1p activity, comprising a CDC4p, CDC34p, and a SKP1p polypeptide, and either a CDC53p or CUL1p polypeptide. In this assay the CDC4p, CDC34p, and SKP1p polypeptide are either a yeast polypeptide or a polypeptide from another species, and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a yeast polypeptide and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a polypeptide from another species. A method is further provided for identifying a compound that affects the ability of a CDC4p, a SKP1p, a CDC34p, and a CDC53p or a CUL1p to ubiquitinate a substrate. The method includes contacting a sample comprising a CDC4p, a SKP1p, a CDC34p, and a CDC53p or CUL1p, with the compound under conditions sufficient to allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a substrate for ubiquitination. The ability of the CDC4p, the SKP1p, the CDC34p, and the CDC53p or CUL1p, to ubiquitinate the substrate is measured. A method is also provided of identifying a polypeptide having a function of a CDC4 subunit of SCF. A method is further provided for identifying a polypeptide as a substrate for a ubiquitination reaction.
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