Nonoverlapping regions of Bdf1 are required for phosphorylation and TFIID binding. (A) Recombinant GST-Bdf1 proteins carrying the indicated amino acids (numbers within parentheses) were expressed in bacteria and purified (top panel). Some proteolysis of the C-terminal regions of Bdf1 was observed, but intact fusion proteins are marked with asterisks. The proteins were immobilized on glutathione-agarose beads and incubated with yeast whole-cell extracts from a strain containing an HA epitope-tagged Taf7. After washing, the beads were tested for phosphorylation of the GST-Bdf1 proteins (middle panel). Binding to TFIID was assayed by immunoblotting for the HA tag on Taf7 (bottom panel). Similar TFIID binding results were obtained when the blots were probed for Taf1 and Taf5 (data not shown). (B) Deletion analysis results from panel A are summarized schematically. The bromodomains are shaded gray, while the acidic C-terminal region is shown in black.

Bdf1 is phosphorylated on serines. (A) The protein sequences of the Bdf1 phosphorylation regions are shown below a schematic diagram of Bdf1. BD1 and BD2 designate the two bromodomains. (B) Phospho-amino acid analysis of phosphorylated Bdf1. GST-Bdf1 derivatives containing the CPR (amino acids 454 to 686; lane 1) or the IPR (amino acids 1 to 29 and 421 to 452; lane 2) were analyzed as described in the text. The left panel is an autoradiogram showing the labeled amino acids, while the right side shows ninhydrin staining of the phosphorylated amino acid markers.

Bdf1 phosphorylated regions are required for function in vivo. A bdf1Δ bdf2Δ strain carrying the BDF1 gene on a URA3 plasmid (YSB529) was transformed with an empty TRP1 vector or TRP1-marked plasmids expressing full-length Bdf1, the ΔIPR mutant (lacking amino acids 441 to 452), or a ΔCPR deletion mutant (lacking amino acids 627 to 686). Growth of the transformants is shown before (control plate) and after 5-FOA selection for loss of the wild-type BDF1 plasmid. Efficient expression of the deletion mutants was demonstrated by immunoblotting (data not shown and Fig. 4 and 5).

Analysis of Bdf1 IPR point mutants. (A) Recombinant GST-Bdf1 derivatives were immobilized on glutathione-agarose beads, exposed to yeast extract, and assayed for phosphorylation. The indicated point mutations in IPR serines were analyzed. Note that all derivatives tested here lacked the CPR (aa 626 to 686). The top panel shows an autoradiograph of phosphorylated proteins, while the bottom panel shows a Coomassie-stained gel of the same proteins. The bacterially produced GST-Bdf1 suffers from some proteolysis, but the full-length protein is marked with an asterisk. (B) The indicated Bdf1 IPR mutants were tested for complementation in a bdf1Δ bdf2Δ strain (YSB529) by plasmid shuffling as described in the legend for Fig. 3. Increasing dilutions (top to bottom) of cells were spotted on the indicated plates. The third panel shows cells before selection on 5-FOA medium for loss of a wild-type BDF1 plasmid. The bottom panel is an immunoblot showing expression of the mutants (which are HA tagged) in cells before 5-FOA selection. The top two panels show cells selected for removal of the wild-type BDF1 plasmid, at both 30 and 37°C. Note that, in contrast to those in panel A, these derivatives all contain the full-length CPR.

Finer mapping of the Bdf1 CPR. (A) Recombinant GST-Bdf1 derivatives were immobilized on glutathione-agarose beads, exposed to yeast extract, and assayed for phosphorylation. The indicated deletion mutations of the CPR were analyzed. Note that all derivatives tested here lacked the IPR (aa 441 to 452). Full-length proteins are marked with an asterisk. (B) The indicated Bdf1 CPR deletion mutants were tested for complementation in a bdf1Δ bdf2Δ strain (YSB529) by plasmid shuffling as described in the legend for Fig. 4B. The bottom panel shows anti-HA blotting for the mutants in cells before 5-FOA selection. Note that these mutants all contain the IPR.

Bdf1 binds to protein kinase CK2. (A) Proteins were purified using a C-terminal TAP-tagged Bdf1 (Bdf1-TAP). An untagged strain (Bdf1) was used as a control. Copurifying bands were identified by mass spectroscopy. CK2 subunits are designated. The major band around 70 kDa is the heat shock 70 protein Ssb1, while the band around 55 kDa is Imd3. Both of these proteins are frequent contaminants in TAPs (7). (B) Bdf1 association with CK2 requires the phosphorylated regions. GST, GST-Bdf1, and the GST-Bdf1 mutant lacking the IPR and CPR were incubated with whole-cell extracts from strains containing HA-tagged Taf7 (YSB892) or no tag (YSB634). After binding and washing, proteins bound to the glutathione-agarose beads were analyzed by SDS-PAGE and immunoblotting for the HA tag. Input extract from YSB892 is shown in the first lane.

CK2 phosphorylates Bdf1. Extracts were made from yeast strains containing mutant CK2 subunits (cka2^ts and cka1^ts; second and fourth lanes) or from their isogenic wild-type CK2 parent strains (CKA2 and CKA1; first and third lanes). Cells for extracts were grown at the permissive temperature. In the top two panels, endogenous Bdf1 was immunoprecipitated with anti-Bdf1 antibody (α-Bdf1 IP), and pellets were phosphorylated with either radioactive ATP or GTP. Anti-Bdf1 immunoblotting showed that all four extracts contained equal amounts of Bdf1 (data not shown). In the bottom four panels, recombinant GST-Bdf1, GST-Bdf2, or GST-CTD was incubated in the extracts, repurified with glutathione-agarose beads, and then phosphorylated with either [γ-³²P]ATP or [γ-³²P]GTP.

hTAF1 can be phosphorylated by CK2. (A) Schematic representation of the alignment between the Bdf1 protein, yeast Taf1, and hTAF1. Boundaries of the NTK and CTK domains (vertical stripes and black, respectively) and the bromodomains (gray boxes) are shown. (B) Recombinant GST-Bdf1, GST-NTK (hTAF1 N-terminal kinase domain; amino acids 1 to 393), or GST-CTK (hTAF1 CTK domain; amino acids 1404 to 1872) was expressed in E. coli. Purified proteins were incubated with yeast whole-cell extracts from wild-type CKA2 or cka2-8^ts mutant strains or in a mock reaction mixture not containing any yeast extract. Proteins were repurified and assayed for phosphorylation as described for Bdf1 in the legend for Fig. 7. The top panel shows an autoradiograph of the phosphorylated proteins, while the bottom panel shows a Coomassie-stained gel of the recombinant proteins.