Identification and characterization of individual cyclin-dependent kinase complexes from Saccharomyces cerevisiae

In S. cerevisiae, regulation of cell cycle progression is known to be carri ed out by a single cyclin-dependent kinase homologue, Cdc28p, acting at dif ferent stages of the cell cycle in association with various cyclins and oth er regulatory subunits. However, a still unsolved problem is the identifica tion of the physiologically relevant substrates of the different Cdc28p kin ase complexes which participate in this regulation. Purification and charac terization of the subunit composition and enzymological properties of these Cdc28p complexes would therefore contribute substantially to our understan ding of the molecular mechanisms controlling the cell cycle. We have used a combination of ammonium sulphate fractionation. nickel nitrilotriacetate a ffinity purification, ATP-Sepharose affinity chromatography and Resource Q ion exchange chromatography to purify two different Cdc28p kinase complexes . Using specific db deletion mutants and plasmid or genomic HA epitope-tagg ed CLBs, we show that one of these complexes is composed almost exclusively (93% or greater) of Clb2p-Cdc28p. whereas the other is mainly (75% or grea ter) Clb3p-Cdc28p. These procedures provide the basis for the analysis of r egulatory, enzymatic and functional properties of individual Cdc28p kinase complexes. Copyright (C) 1999 John Wiley & Sons, Ltd.