Figure 4. Complementation of PP1-mutated yeast by SmPP1.

(A) SmPP1 expression in yeast. Western blot analysis of SmPP1 from wild-type Schizosaccharomyces pombe expressing empty vector (lane 1), Rep1–SpPP1 Dis2 (lane 2) and HA-tagged Rep1–SmPP1 (lane 3). The extracts of each transformant were immunoblotted with anti-HA (upper panel) or anti-SpPP1 (lower panel) antibodies. The arrow indicates the SmPP1 and the arrowhead indicates the SpPP1. (B) Frequencies of rescued yeast cells. The SpPP1-mutated strain was transformed with empty vector (Rep1) or Rep1–SmPP1 or Rep1–SpPP1 vector. Results are means±S.D. for two independent experiments performed with two different clones. In this yeast model, transformation with empty vector yielded up to 44.4% of abnormal mitosis. In the case of Dis2 and SmPP1 transformations, the percentage of cells with abnormal mitosis was 16.66% and 2.5% respectively. For clarity, we considered that the percentage of abnormal cells obtained with the empty vector alone is 100%. The percentages of abnormal cells obtained after transformation with Dis2 or SmPP1 vectors were calculated using the following formula: X=(percentage of abnormal cells obtained with either Dis2 or SmPP1 vector/percentage of abnormal cells obtained with empty vector alone)×100, thus allowing us to present data as frequencies of rescued cells (100−X%). The inset shows DAPI staining microscopy of Schizosaccharomyces pombe. Left: SpPP1-null single yeast showing condensed chromosomes typical of mitosis deficiency; right: yeast rescued with SmPP1, where the chromosomes are found at both ends of the cell.