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. 2014 Dec 1;290(3):1332–1347. doi: 10.1074/jbc.M114.597948

FIGURE 3.

FIGURE 3.

Drosophila DDK complex can rescue a temperature-sensitive growth defect in cdc7-90 yeast. A, growth assay showing that co-expression of D. melanogaster Cdc7 and Chiffon restores growth at the restrictive temperature (30 °C) in yeast carrying the temperature-sensitive cdc7-90 allele, whereas the expression of DmCdc7 with a mutation in a conserved kinase motif (D269N) does not. cdc7-90 yeast were co-transformed with the indicated plasmid combinations, and yeast strains were plated on selective media and incubated at permissive (23 °C) or restrictive (30 °C) temperatures. B, Western blots verifying the expression of Cdc7 and Chiffon in the cdc7-90 rescue strains shown in A. Cdc7 and Chiffon were expressed as fusion proteins with N-terminal epitope tags as follows: HA, Cdc7; FLAG, Chiffon. Histone H3 was analyzed as a loading control. C, growth assay showing that co-expression of D. melanogaster CG5790 and Chiffon does not restore growth of yeast carrying the temperature-sensitive cdc7-90 allele at the restrictive temperature (30 °C), as described in A. Plasmid combinations contained within each yeast strain are as indicated. D, Western blots verifying the expression of Cdc7, CG5790, and Chiffon in the cdc7-90 rescue strains shown in C, as described in B. CG5790 was expressed as a fusion protein with an N-terminal HA epitope tag. E, HA-CG5790 co-immunoprecipitates Chiffon in lysates from cdc7-90 yeast strains ectopically expressing epitope-tagged CG5790 and Chiffon as described in C. Lysates were incubated with anti-HA affinity resin, and co-immunoprecipitating proteins were analyzed by SDS-PAGE and Western blotting. F, schematic comparing the locations of conserved N, M, and C domains in the Dbf4 orthologs ASK, Chiffon, and an N-terminal Chiffon truncation, ChiffonN (amino acids 1–400). The percent identity of the N, M, and C domains in human ASK and Chiffon is indicated. G, growth assay showing that co-expression of DmCdc7 with either full-length Chiffon (ChiffonFL) or ChiffonN restores growth at the restrictive temperature (30 °C) in yeast carrying the temperature-sensitive cdc7-90 allele as described in A. H, Western blots verifying the expression of Cdc7 and full-length and truncated Chiffon in the cdc7-90 rescue strains shown in G, as described in B.