Figure 1.

Characterization of the condensin protein complex. (A) CLUSTALW alignment of the condensin subunits from different organisms. Arabidopsis thaliana and human sequences are from GenBank. (B) Immunoprecipitation of condensin throughout the cell cycle. Strains YPH499bp (mock) and YP499bp5 were arrested with α-factor (G1, 100% of cells arrested), hydroxyurea (S, 84% arrest), and nocadazole (M, 96% arrest). 12CA5 immunoprecipitates from the corresponding protein extracts are shown for YPH499bp5. Smc2p, Smc4p, Brn1p, and Ycs4p are detected with the corresponding rabbit polyclonal antibodies. The precipitates were probed with antibodies against chromatin proteins, including Hmo1, Smc1p, Mcd1p, and Top2p, but no positive signal was detected (data not shown). (C) Smc2p and Smc4p form an obligatory complex. Asynchronous cells of YPH499bp2 were analyzed by immunoprecipitation. Saturating amount of anti-HA beads were added, allowing quantitative depletion of Smc2p-HA. IN, input; FT, flow-through; WA, wash; and EL, elution. (D) Stoichiometry of non-SMC condensin subunits. Extracts from YPH499bp6, BY4733bp4, and YPH499bp5, grown to equal optical density were analyzed by anti-HA immunoprecipitation. Immunoprecipitates were probed with anti-HA, anti-Smc2p, and anti-Smc4p antibodies. (E) Immunoaffinity purification of condensin complex. 10 g of the YPH499bp5 cell was subjected to the modified immunoprecipitation protocol, followed by the peptide elution with AS4 (GYPYDVPDYAG) (0.5 mg/ml) in place of SDS. 10 g of YPH499bp cells was processed identically for the mock purification. The silver-stained gel (4–12% PAGE) shows stoichiometry of condensin subunits. Asterisks indicate positions of the condensin subunit bands on the gel. Proteins were identified by Western blot with the corresponding antibodies. Smc2p (predicted molecular mass 134 kD) and Ycs4p (133 kD) comigrate.