Abstract:
:The anaphase-promoting complex (APC) is a central regulator of the eukaryotic cell cycle and functions as an E3 ubiquitin protein ligase to catalyze the ubiquitination of a number of cell cycle regulatory proteins. The APC contains at least 13 subunits in addition to two activator subunits, Cdc20 and Cdh1, that associate with the APC in a cell cycle-dependent manner. This chapter describes methods for preparation and assay of the APC from Saccharomyces cerevisiae. Highly active APC is purified from cells expressing Cdc16 fused with a tandem affinity purification (TAP) tag. Enzymatically active APC is achieved upon addition of recombinant Cdc20 or Cdh1 together with E1, Ubc4, ATP, and ubiquitin. Activity assays toward several endogenous substrates, including Clb2 and Pds1, are described. In addition, methods for observation of APC-coactivator and APC-substrate complexes by native gel electrophoresis are described.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Passmore LA,Barford D,Harper JWdoi
10.1016/S0076-6879(05)98017-8subject
Has Abstractpub_date
2005-01-01 00:00:00pages
195-219eissn
0076-6879issn
1557-7988pii
S0076-6879(05)98017-8journal_volume
398pub_type
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