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Fig. 7. Orc1p but not Orc2p binds to HP1. (A) GST pull-down experiment performed by incubating 2 µg of either GST or GST-HP1 immobilized on glutathione-agarose beads with in vitro translated [³⁵S]-labeled Orc1p or Orc2p. After binding at 4°C, the beads were extensively washed and bound proteins were loaded onto a 10% acrylamide-SDS gel. The upper panel shows the autoradiograph; the lower panel shows the gel after staining with Coomassie blue. The input lanes contain the labeled proteins prior to binding. (B) Quantification of the GST pull-down experiment from A. The amount of radioactivity bound to the beads is indicated as a percentage of the input material. (C) HP1 binds to a region of Orc1p encompassing amino acids 151-269. The indicated [³⁵S]-labeled deletion mutants of Orc1p were incubated with either GST or GST-HP1 and processed as described in A. (D) Quantification of the GST pull-down experiment of panel C. (E) Visualization of FRET in human HeLa cells. The plasmids indicated on top of each column were transfected in asynchronous HeLa cells; individual transfected cells were visualized by excitation at 480 nm and collection at 520 nm, showing EGFP fluorescence after direct EGFP excitation (panels in row a), and by excitation at 350 nm and collection at 520 nm, showing EGFP fluorescence after BFP excitation, indicating FRET (panels in row b). (F) Quantification of FRET between EGFP-HP1 and BFP-Orc1. Fluorescent emission at 520 nm from individual cells transfected with the indicated constructs was recorded after excitation at 350 or 480 nm, and integrated intensities over the whole cell were evaluated. The plotted values (indicated by dots) represent the ratio between these two measurements: higher values indicate more efficient resonant energy transfer between BFP and EGFP. Ten consecutively analyzed cells were considered for each transfection; both their individual fluorescence ratios and their percentile box plot distributions are shown. In each box, the horizontal lines from top to bottom mark the 10th, 25th, 75th and 90th percentiles. Cells transfected with pEGFP-HP1 and pBFP-Orc1 plasmids showed FRET between the two fluorescent proteins that was dependent on the presence of both the HP1 and Orc1p moieties, thus indicating binding between these two proteins in vivo.

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