Figure 3
Subcellular localization of SCFPop subunits (3A) Pop1p, Pop2p, Pcu1p, Psh1p, and Pip1p were mildly overexpressed in wild-type cells as N-terminally GFP-tagged proteins from pRep81 for 20 h. Cells were fixed in para-formaldehyde and mounted onto poly-lysine coated cover slips. Nuclei were counterstained with DAPI. Fluorescence images were obtained with a Spot CCD camera mounted onto a Nikon E600 epifluorescence microscope. (3B) Immunoblot of cells genetically modified to contain Pop1p or Pop2p tagged with 13 C-terminal Myc epitopes at the endogenous genomic loci. (3C) Differential subcellular localization of endogenous Pop1p and Pop2p. The cells described in (B) were fixed in para-formaldehyde and processed for immunostaining with Myc antibodies as described [25]. Wild-type cells not containing any Myc-tagged alleles were used as specificity controls. DAPI stained cell nuclei are indicated. (3D) The epitope-modified cells described above were fractionated into nuclear and cytoplasmic proteins as described in materials and methods. Equal proportions of both fractions were separated by SDS PAGE and probed with Myc antibodies to detect Pop1p and Pop2p (top panel). Blots were reprobed with antibodies directed against cytoplasmic tubulin (middle panel) and nuclear PCNA (bottom panel) to estimate the efficiency of the fractionation protocol. (3E) Cells harboring 13Myc epitope-tagged Pop1p in a pop2 deficient background (pop1-13myc Δpop2) or 13Myc-tagged Pop2p in a pop1 mutant (pop2-13myc Δpop1) were fixed and processed for immunostaining with Myc antibodies. As reference, the strains described above, which contain Myc-tagged Pop1p or Pop2p in a wild-type background are shown.