Juanes MA , Khoueiry R , Kupka T , Castro A , Mudrak I , Ogris E , Lorca T , Piatti S .

	Figure 2. Yeast Igo1 induces mitotic entry in Xenopus egg extracts.A. Human hyperactive Gwl (Gwl-K72M) was used to phosphorylate in vitro GST-Igo1 and GST-Igo1S64A purified from bacterial cells. B. Bacterially purified GST-Igo1 or GST-Igo1S64A were phosphorylated in vitro by human Gwl and added at the indicated concentrations to PP2ACdc55 complexes affinity-purified from yeast cells expressing HA-tagged Cdc55. The “no tag” control corresponds to the activity of an anti-HA affinity purification from cells expressing untagged Cdc55. C. Igo1 or Igo1S64A previously phosphorylated in vitro by Gwl were added to Xenopus egg interphase extracts at time 0. At the indicated time points aliquots of extracts were frozen in liquid nitrogen and subsequently analysed by western blot for Gwl and Cdc25 phosphorylation (causing a mobility shift in mitosis) and Cdk1 phosphorylation on Tyr15. Cdk1 kinase activity was measured using histone H1 as substrate. D. GST-Igo1 and GST-Igo1S64A were pulled down to assess their association with A, B55 and C subunit of PP2A by western analysis.
	Figure 3. Deletion of RIM15 or IGO1 and IGO2 delays mitotic entry under temperature stress.Cycling (cyc) cultures of wild type, rim15Δ and igo1Δ igo2Δ cells were arrested in G1 by α-factor and released in fresh medium at 38°C (A) or 16°C (B). At the indicated time points cells were collected for FACS analysis of DNA contents (not shown), kinetics of budding, spindle assembly/elongation and nuclear division (graphs), as well as to prepare protein extracts for western blot analysis of Clb2 and Cdc5 levels and for Clb2/Cdk1 kinase activity using histone H1 as substrate. Pgk1 was used as loading control.
	Figure 4. Igo proteins are required for full PP2ACdc55 activity and timely Cdk1-Tyr19 and Mih1 dephosphorylation.A–B. HA-Cdc55 expressed from the TPI1 promoter was immunoprecipitated from lysates of wild type (wt) or igo1Δ igo2Δ cells. Immunoprecipitates were analysed by western blot for the presence of HA-Cdc55, Pph21 and Tpd3 (A) and in parallel assayed for phosphatase activity using phosphorylase a (n = 12) and histone H1 (n = 5) as substrates (B). Statistical significance of differences was assesses by Student's t-test (** p<0.01). C. HA-Cdc55 expressed at endogenous levels from the CDC55 promoter was immunoprecipitated from lysates of wild type (wt) or igo1Δ igo2Δ cells. Immunoprecipitates were analysed by western blot for the presence of HA-Cdc55, Pph21 and Tpd3. D. Cells with the indicated genotypes were arrested in G1 by α-factor and released for 2 hours in the presence of nocodazole. The levels of Tyr19-phosphorylated Cdk1 (Cdk1-Y19-P) were measured by western blot analysis. E. Total lysates of wild type, rim15Δ, igo1Δ igo2Δ and cdc55Δ cells expressing HA-tagged Mih1 (Mih1-HA3) and logarithmically growing at 25°C were analysed by western blot with anti-HA antibodies. F. Logarithmically growing (cyc) cultures of the same strains as in (A) were arrested in G1 by alpha factor and released in the cell cycle at 38°C to analyse at the indicated time points the electrophoretic mobility of Mih1-HA3 by western blot. Cell samples were also collected at the indicated time points to measure DNA contents by FACS analysis (not shown), as well as kinetics of budding, spindle assembly/elongation and nuclear division (graphs).
	Figure 5. Deletion of RIM15 or IGO1 and IGO2 affects the positive feedback loop for Cdk1 activation.A–B. Serial dilutions of strains with the indicated genotypes were spotted on YEPD plates and incubated for 48 hours at the indicated temperatures. C. Cycling (cyc) cultures of wild type, igo1Δ igo2Δ and igo1Δ igo2Δ swe1Δ cells were arrested in G1 by α-factor and released in fresh medium at 38°C. At the indicated time points cells were collected for FACS analysis of DNA contents (not shown), kinetics of budding, spindle assembly/elongation and nuclear division (graphs), as well as to make protein extracts for western blot analysis of Clb2 and Cdc5 levels. Pgk1 was used as loading control. D. Serial dilutions of strains with the indicated genotypes were spotted on YPD and YPGal plates and incubated at 25°C for 2 days.
	Figure 6. Igo1 and Igo2 control the subcellular localization of PP2ACdc55 independently of Zds proteins.A. Localization of HA3-Cdc55 was analysed by indirect immunofluorescence with anti-HA antibodies after formaldehyde fixation of cycling cultures of wild type, rim15Δ igo1Δ igo2Δand igo1Δ igo2Δ swe1Δcells. Cells were scored according to their cell cycle stage: unbudded (G1), budded mononucleated (S, G2, early M) and budded binucleated (late M). At least 15 cells were scored for each class. B. Interaction between Zds1-Pk6 and HA3-Cdc55 in the presence or absence of Igo proteins (left panel) and interaction between Igo1-Pk3 and HA3-Cdc55 in the presence or absence of Zds proteins (right panel) was assessed by immunoprecipitation with anti-Pk antibodies followed by western blot analysis with anti-HA and anti-Pk antibodies. The asterisk marks a protein aspecifically recognized by the anti-Pk antibodies in the total extracts (input) and that co-migrates with Zds1-Pk6.
	Figure 7. Model.The mitotic CDK Clb2-Cdk1 is part of a positive feedback loop where it contributes to its own activation by promoting a transcriptional mitotic program for its own expression and that of the polo kinase Cdc5. Although Swe1 phosphorylation by Clb2-Cdk1 promotes Tyr19 phosphorylation, and thereby inhibition, of Cdk1 (not depicted), it primes further Swe1 phosphorylations by several other kinases, including Cdc5, that finally target Swe1 to degradation. Initial Swe1 phosphorylation by Cdk1 is counteracted by the PP2ACdc55 phosphatase, which also dephosphorylates and activates Mih1, thereby participating in the positive feedback loop for Cdk1 activation. The endosulfines Igo1 and Igo2, upon phosphorylation by Rim15, bind to PP2ACdc55 and contribute to their activation and nuclear export. The observation that SWE1 deletion restores normal localization of Cdc55 in igo1Δ igo2Δ mutant cells suggests that Swe1 and/or Clb2-Cdk1 might regulate the nuclear export of Cdc55 through an unknown mechanism. See text for further details.
	Figure 1. Igo1 interacts with PP2ACdc55 upon Ser64 phosphorylation in lateS/G2 phase of the cell cycle. A. Primary sequence alignment of endosulfines from different species around the serine phosphorylated by Greatwall/Rim15. B. Cell extracts from wild type and rim15Δ cells expressing HA3-Cdc55 and Igo1-Pk3 as only sources of Cdc55 and Igo1 were subjected to immunoprecipitation with anti-Pk antibodies. The amount of HA3-Cdc55, Pph21, Tap42 and Rts3 co-immunoprecipitating with Igo1-Pk3 was assessed by western blot analysis. An extract from an Igo1-untagged strain was used as negative control (no tag). The asterisk marks the IgG in the IPs. C. Co-immunoprecipitations of HA3-Cdc55 with either wild type Igo1-myc8 or Igo1-S64A-myc8, in the presence or absence of RIM15. D–E. Cycling (cyc) cultures of cells expressing HA3-Cdc55 and Igo1-Pk3 was arrested in G1 by α-factor and released in fresh medium at 25°C. At different time points after release cell samples were collected to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies (left, IP:αPk), the protein levels of HA3-Cdc55, Igo1-Pk3, Clb2, Cdc5 (left, Input), as well as the kinetics of budding, DNA replication (by FACS, data not shown), spindle formation/elongation and nuclear division (right graph). Pgk1 was used as loading control for the western blot. F. Cycling (cyc) cells were arrested in G1 by alpha factor or in S phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc) to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies. G. A cycling (cyc) culture of cells expressing Igo1-myc8 was arrested in G1 by alpha factor and released into the cell cycle at 25°C. At the indicated time points cell samples were collected for FACS analysis of DNA contents (not shown) and to prepare TCA protein extracts that were run on precast Phos-tag gels to visualize the phosphorylation of Igo1 by mobility shift. Extracts from rim15Δ and Igo1-S64A-myc8 cells were loaded as controls. Note that alpha factor induces an additional mobility shift likely corresponding to additional phosphorylations that are quickly lost upon cell cycle entry.

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