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First published online 19 February 2003
doi: 10.1242/jcs.00314

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Polo boxes form a single functional domain that mediates interactions with multiple proteins in fission yeast polo kinase

Nicola Reynolds^* and Hiroyuki Ohkura

The Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK
^* Present address: MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK

View larger version (66K): [in a new window]   Fig. 1. Conservation of amino acid sequences among polo kinase homologues and mutations of S. pombe Plo1 created in this study. Residues shaded in dark or light blue represent identical residues among all or most of these polo kinase homologues respectively. Mutations or truncations created by site-directed mutagenesis are marked in red and sites of mutations created by random mutagenesis are marked in green.

View larger version (82K): [in a new window]   Fig. 2. Polo boxes are required for interference of bipolar spindle formation, but not for induction of septation upon overexpression of Plo1. Wild-type cells carrying plo1 mutants in pREP1 under the control of the thiamine repressible nmt1 promoter were grown in the absence of thiamine. The cells were fixed and stained with DAPI, which highlights DNA, cell outlines and septa. No defects were apparent with any of the plasmids in the presence of thiamine. Bar, 10 µm. (A) Empty vector, pREP1 has no effect on cell growth. (B) Overexpression of wildtype plo1 (pREP1plo1+) leads to mitotic arrest and cells with extra septa. (C,D) Overexpression of mutants in the polo boxes (plo1W497F,plo1.1-583 are shown here as examples) resulted in uncontrolled formation of septa but no mitotically arrested cells. (E) Overexpression of a catalytic inactive plo1 (plo1K69R) results only in mitotically arrested cells with highly overcondensed chromosomes with no cells forming extra septa. (F) Overexpression of a catalytically inactive polo-box mutant plo1K69R YQL508AAA. No abnormalities can be seen. (G-I) Monopolar spindles were associated with overcondensed chromosomes in cells overexpressing plo1K69R. The SPB component -tubulin (G), Sad1 (H) and DNA (I). (J-L) Quantification of cytological defects seen upon overproduction of wild-type Plo1 (J), Plo1 with a polo box mutation (Plo1DHK625AAA; K) or kinase inactive Plo1 (Plo1K69R; L) in wild-type or mad2 deletion strains. Cells were observed by DAPI staining after the removal of thiamine to induce expression from the nmt1 promoter. Mitotic arrest caused by overproduction of inactive Plo1 is abolished by a mutation in the spindle checkpoint, mad2.

View larger version (49K): [in a new window]   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.

View larger version (79K): [in a new window]   Fig. 3. Mutations in the polo boxes disrupt Plo1 function in vivo. Diploid strains were constructed in which one copy of plo1+ is disrupted by the his3+ gene and plo1 mutant gene marked with the ura4+ gene is integrated at one of the leu1 loci. The diploids were sporulated and germinated under the selective condition (—His —Ura), which allows the growth of only the plo1 disruptant expressing mutant plo1 genes. Cells were fixed and stained with DAPI. (A) plo1 disruptant cells expressing plo1YQL508AAA. (B) plo1 disruptant cells expressing plo1DHK625AAA. Both types of cells exhibit similar cytological defects to the plo1 disruptant. These defects include mitotic arrest with overcondensed chromosomes (left panels), disorganised septum (middle) and a failure of septation (right). Round cells seen in B are ungerminated spores. Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 5. Polo boxes play a crucial role in determining the protein interactions. (A) A schematic diagram of the experimental design to identify plo1 mutants that disrupt a subset of interactions (see Materials and Methods for detail). The plo1+ gene is randomly mutagenised by an error-prone PCR (about one mutation per kb). Yeast strain L40 was co-transformed with the PCR product and a gapped bait vector. Short sequences shared between the ends of the bait vector and the PCR products allow gap repair in vivo to recreate bait plasmids with various plo1 mutations. Each strain carrying mutagenised plo1 bait constructs was mated with Y187 strains carrying prey plasmids. The two-hybrid interaction was assessed by expression of the HIS3 reporter gene. Plasmids were isolated from strains of interest, re-transformed to confirm the interaction pattern by expression of another reporter lacZ, and sequenced to determine the mutation site. (B) Sequence analysis of some Plo1 mutants that show differential interactions. Most of the mutations mapped in the polo boxes, which suggests that polo boxes are crucial for determining protein interaction. — (no or marginal interaction) < + (weak) < ++ (intermediate) < +++ (interaction at same levels as wild-type Plo1).

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