Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 12 (2010): 886-893, doi:10.1038/ncb2092. ; When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5, 6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found that PP2A-B55α functions downstream of Cdk1 inactivation. PP2A-B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate histone H1 and it was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor Importin β1, and RNAi depletion of Importin β1 delayed mitotic exit synergistically with PP2A-B55α. This demonstrates that PP2A-B55α and Importin β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. ; This work was supported by SNF research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to DWG, and a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to DWG, a Roche Ph.D. fellowship to MHAS, and a Mueller fellowship of the Molecular Life Sciences Ph.D. program Zurich to MH. MH and MHAS are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. VJ and JG were supported by grants of the ‘Geconcerteerde OnderzoeksActies’ of the Flemish government, the ‘Interuniversitary Attraction Poles’ of the Belgian Science Policy P6/28 and the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’. AIL is a Wellcome Trust Principal Research Fellow. AAH acknowledges funding by the Max Planck Society, the EU-FP6 integrated project MitoCheck, and the BMBF grant DiGtoP [01GS0859]. Work in the groups of KM and JMP was supported by the EU-FP6 integrated project MitoCheck, Boehringer Ingelheim and by the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), by MeioSys within the Seventh Framework Programme of the European Commission, and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF).