Fig. 4. The non-catalytic region is sufficient for SPB localisation. (A-C) Autofluorescence of GFP-tagged Plo1 mutant proteins (green) and immunostaining of the SPB component Sad1 (red) and DNA (blue) are shown. Co-localisation of GFP-Plo1 and Sad1 signals is seen in yellow in the merge panel. (A) GFP-Plo1⁺ localises to the SPB in late G2 or early in mitosis before SPB separation (cell on right) and remains there throughout metaphase to anaphase (cell in middle), gradually becoming weaker late in anaphase (cell on left). Newly divided cells do not have GFP signals on SPB. Localisation to the actin ring or mitotic spindle is not detected using this construct. Bar, 10 µm. (B) GFP-Plo1K69R is indistinguishable from GFP-Plo1. (C) GFP-Plo1DHK625AAA uniformly localises to the cytoplasm throughout the cell cycle. This localisation pattern was common to all polo box point mutants and truncations. The integrity of all polo boxes is therefore required for localisation to SPBs. (D) The non-catalytic domain of Plo1 is sufficient for cell-cycle-regulated localisation to the SPBs. Small early G2 cells expressing GFP-Plo1.313-683 were collected by centrifugal elutriation and then cultured in minimal media at 30°C. Samples were taken every 20 minutes for examination of GFP signals on SPBs (one or two) under a fluorescent microscope. In the upper panel, percentages of cells with no GFP foci, one foci and two foci were represented by open circles, closed squares and open triangles, respectively. The septation index is shown in the lower panel. (E) Expression of GFP-tagged mutant Plo1 proteins assayed by western blotting using a Plo1 antibody. The amounts of the GFP-tagged proteins are comparable with endogenous Plo1 protein except Plo1.1-483 and Plo1.473-683, which were not detectable.

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