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The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase

Gilles Crevel, Helen Bates, Hella Huikeshoven and Sue Cotterill^*

Department of Biochemistry and Immunology, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK
These authors contributed equally to this paper

View larger version (74K): [in a new window]   Fig. 1. Predicted amino acid sequence of Dpit47, human TTC4 and S. cerevisiae CNS1. Shaded boxes indicate regions of identity. The TPR domain region is indicated below the sequence. D.m., Drosophila melanogaster; H.s., Homo sapiens; S.c., Saccharomyces cerevisiae; TPR, tetratrico peptide repeat.

View larger version (12K): [in a new window]   Fig. 2. Co-immunoprecipitation of Dpit47 and DNA polymerase . Drosophila embryo crude extract was subjected to immunoprecipitation using a polyclonal antibody against Dpit47 cross-linked to protein A sepharose. The immunocomplexes were washed extensively and then eluted with increasing salt concentrations and SDS as indicated in the figure. The top panel represents the elution profile of DNA polymerase (polp180), and the bottom represents that of the Dpit47 in the same experiment. c, control immunoprecipitation using an unrelated antibody. 1 and 2, immunoprecipitation using anti-Dpit47 antibody (duplicate experiments are shown for each immunoprecipitate).

View larger version (58K): [in a new window]   Fig. 3. Dpit47 forms a complex with Hsp90 and Hsp70. The pellets from two independent Dpit47 immunoprecipitations that had been washed in 500 mM NaCl were analysed by electrophoresis on SDS-PAGE and staining with sensitive Coomassie (Brilliant blue G colloidal). *, indicates the IgG heavy band. Sizes of molecular weight markers are shown to the left of the gel.

View larger version (34K): [in a new window]   Fig. 4. (A) The interaction between Dpit47 and DNA polymerase is stabilised by geldanamycin (g) and destabilised by ATP. Dpit47 was immunoprecipitated from crude embryo extract with anti-Dpit47 antibody cross-linked to protein A sepharose and eluted with 250 mM Nacl as in Fig. 2. The eluates were analysed on western blots using polyclonal anti-DNA polymerase antibody. (a) Dpit47 immunoprecipitates were carried out either in the presence (+g) or absence (-) of 1 µg/ml geldanamycin as described in materials and methods. (b) Dpit47 immunoprecipitates were carried out as above either in the presence of 1 µg/ml geldanamycin (+g) or 1 mM ATP (+ATP). (B) The interaction between Dpit47 and Hsp70 is destabilised by ATP. The 500 mM washed pellets of the immunoprecipitation as described above were analysed on SDS-PAGE and stained with Coomassie blue. The bands corresponding to Hsp90 Hsp70 and Dpit47 are shown. a, no treatment of the immunoprecipitate; b, treatment with geldanamycin; c, treatment with ATP.

View larger version (24K): [in a new window]   Fig. 5. Western blots show the abundance of Dpit47 throughout the developmental stages. Embryonic development is represented by the indicated age of embryos from 0 to 20 hours of development. L, larvae; P, pupae; M, males; F, females. Equal loading of samples was confirmed using a Drosophila anti-importin alpha-3 antibody (imp3) (Mathe et al., 2000).

View larger version (37K): [in a new window]   Fig. 6. DNA polymerase is associated with Dpit47 throughout development and in proliferating cell culture but not in quiescent cells. (a) A comparison of the amount of DNA polymerase associated with Dpit47 complexes immunoprecipitated at different stages of the life cycle from 0 to 24 hours as indicated. The amounts of DNA polymerase in each extract is indicated (loading controls). In each case equal amounts of the immunoprecipitated Dpit47 was loaded as determined by western blotting. (b) A comparison of the amount of DNA polymerase associated with Dpit47 complexes immunoprecipitated from Drosophila embryo extract (E) and exponentially growing S2 cells (C). The two panels on the left are western blots showing the amounts of DNA polymerase and Dpit47 in crude extracts from Drosophila embryos and S2 cells. The panel on the top right shows the amount of DNA polymerase (as determined by western blot) immunoprecipitated with anti-Dpit47 antibody from each extract. The bottom right panel has been included to show that equal amounts of Dpit47 (as analysed by coomassie staining) were analysed for each extract. (c) A comparison of the amount of DNA polymerase associated with Dpit47 complexes immunoprecipitated from exponentially growing (D) or quiescent (Q) S2 cells. The two panels on the left are western blots showing the amounts of DNA polymerase and Dpit47 in crude extracts from exponentially growing cells and quiescent cells. The panel on the top right shows the amount of DNA polymerase (western blot) immunoprecipitated with anti Dpit47 antibody for each extract. As above the bottom right panel has been included to show that equal amounts of Dpit47 immunoprecipitate have been compared for each cell type (in this case by western blot).

View larger version (167K): [in a new window]   Fig. 7. Subcellular and cell cycle distribution of Dpit47. (a) Confocal micrographs of Dpit47 stained embryos that have been counterstained with anti- tubulin monoclonal antibodies. (b) Confocals micrographs of Dpit47 stained embryos which have been counterstained with anti-histone monoclonal antibodies. In the merged images in each case the Dpit47 is green whereas the counterstain is red. Stages of the cell cycle are indicated to the left of the panels.

View larger version (37K): [in a new window]   Fig. 8. Cellular fractionation of Dpit47 and DNA polymerase . Drosophila embryo extracts were subjected to salt and detergent fractionation and analysed by western blot using anti Dpit47 and anti-DNA polymerase (pol) antibodies as described in Materials and Methods. Ce, total cellular extract as a control; 50 mM NaCl, cytoplasmic fraction; Triton, protein released on treatment of nucleus with triton, representing the nucleoplasmic fraction; Triton final, last triton wash before salt extractions to show that there is no carry over between fractions; 250 mM NaCl, Triton pellet extracted with 250 mM NaCl and 1% Triton; pellet, remainder after all extractions.

View larger version (32K): [in a new window]   Fig. 9. Analysis of Dpit47 DNA polymerase complexes in the cytoplasm and in the nucleoplasm. Cytoplasmic and nucleoplasmic fraction were subjected to immunoprecipitations using anti-Dpit47 antibody. The DNA polymerase eluted in 250 mM NaCl from two separate experiments fractions was determined by western blots using anti-DNA polymerase antibody. For the cytoplasmic fraction (top panel) the lane ce on the left corresponds to shorter exposure time in order to visualise the band. The same exposure time as the eluted fractions is shown on the right. i and ii, two separate elutions; ce, crude extract control.

View larger version (61K): [in a new window]   Fig. 10. The primase subunit p50 is detected in the complex immunoprecipitated by anti-Dpit47 antibody. Dpit47 immunoprecipitates as described in Materials and Methods were analysed using anti-p180 (polp180) and anti-p50 (polp50) antibodies. In each case two seperate experiments are shown. The lane after the ce contains the last wash of the immunoprecipitate prior to elution to show that there is no carryover between fractions. 250 mM and 500 mM, the 250 mM and 500 mM NaCl elutions from the immunoprecipitate, respectively; ce, crude extract as a control.

View larger version (35K): [in a new window]   Fig. 11. The DNA polymerase complexed with Dpit47 is inactive. DNA polymerase was immunoprecipitated using anti-Dpit47 antibody or monoclonal anti-DNA polymerase antibody. Top panel, DNA polymerase assay: immunoprecipitated DNA polymerase was used to carry out an end filling polymerase assay on a 35 mer as described in Materials and Methods. The reaction was stopped after 5, 15 or 30 minutes. The product of the reaction was analysed on a 15% polyacrylamide gel. Lower panel shows by western blotting the amount of DNA polymerase contained in the Dpit47 (Dpit47 IP) and DNA polymerase (pol IP) immunoprecipitates used in the assay.