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Figure 2. Absence of Cdc55 suppresses the gametogenesis- and quiescence- entry defect of endosulfine mutants.A) Wild-type, igo1Δ igo2Δ, PCLB2CDC55 and igo1Δ igo2Δ PCLB2CDC55, igo1Δ igo2Δ PCLB2CDC55 net1-6Cdk, igo1Δ igo2Δ PCLB2CDC55 NET1, rim15Δ, rim15Δ PCLB2CDC55, rim15Δ PCLB2CDC55 net1-6Cdk, rim15Δ PCLB2CDC55 NET1, rts1Δ and igo1Δ igo2Δ rts1Δ cells were incubated on sporulation plates for 24 hours and number of sporulated (includes monad, dyad, triads/tetrads) and unsporulated cells were counted using a light microscope. B) Wild-type, igo1Δ igo2Δ and igo1Δ igo2Δ PCLB2CDC55 cells were induced to enter meiosis by transferring them to SPM. Pre-meiotic DNA replication in the cultures was assayed by flow cytometry. C) Analysis of expression of Cdc5. Whole-cell extracts of hourly culture in SPM was prepared by TCA method. Protein samples were run on 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-Cdc5 and anti-Cdc28 antibody respectively. D) Wild-type, igo1Δ igo2Δ, cdc55Δ and igo1Δ igo2Δ cdc55Δ cells expressing Hsp26-ha3 were grown to log phase at 30°C, rapamycin (final concentration 200 ng/ml) was added to each culture and samples were collected at indicated times. Total cell extracts were prepared by TCA method. Proteins were run on 12% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-HA and anti-Cdc28 antibodies.
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Figure 3. Igo1 associates and inhibits the phosphatase activity of PP2ACdc55 in a phosphorylation dependent manner.A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from CDC55-TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from CDC55-TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2ACdc55. Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM phosphopeptide (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).
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Figure 4. dhh1Δ and ccr4Δ do not suppress the G0 - and gametogenesis- entry defects of endosulfine mutant cells.A) Sporulation efficiency of igo1Δ igo2Δ, igo1Δ igo2Δ PCLB2CDC55, igo1Δ igo2Δ dhh1Δ and igo1Δ igo2Δ ccr4Δ cells was measured (n = 200). B) Wild type, igo1Δ igo2Δ, cdc55Δ, igo1Δ igo2Δ cdc55Δ, igo1Δ igo2Δ dhh1Δ and igo1Δ igo2Δ ccr4Δ cells expressing HSP26-ha3 were grown to log phase at 30°C. Rapamycin (final concentration 200 ng/ml) was added to the culture and processed as described above. Whole cell extracts were subjected to SDS-PAGE followed by western analysis using anti-HA and anti-Cdc28 antibodies.
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Figure 5. The G0-specific transcription factors Msn2, Msn4 and Gis1 are not required for entry into gametogenesis.A) igo1Δ igo2Δ, msn2Δ msn4Δ gis1Δ, igo1Δ igo2Δ msn2Δ msn4Δ gis1Δ mutant cells were incubated on sporulation plates for 24 hours and sporulation efficiency was measured (n = 200). B) Wild type, igo1Δ igo2Δ and msn2Δ msn4Δ gis1Δ cells carrying pRS316-HSP26-ha3 plasmid were grown to log phase. Rapamycin (200 ng/ml) was added to the culture and total protein extract was prepared from cells at the indicated time points and Western analysis was performed using anti-HA and anti-Cdc28 antibodies.
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Figure 6. Endosulfines are required for transcriptional induction of IME1 caused by transfer of cells to sporulation medium.A) Wild-type and igo1Δ igo2Δ cells were induced to enter meiosis by transferring them to sporulation medium (SPM). Total RNA was prepared from cells after 0, 2, 4, 6 and 8 hours following transfer to SPM and IME1 transcript levels were assayed by quantitative RT-PCR. The IME1 transcript levels were normalized with respect to ACT1 mRNA and expressed relative to normalized IME1 transcript levels in mitotically grown cells. B) Wild-type and igo1Δ igo2Δ cells containing PGAL-ha3-IME1 GAL4-ER were induced to sporulate. Either β-estradiol or ethanol was added to the cultures at t = 0 h. After 24 hours, sporulation efficiency was measured (n = 200). C) Aliquots of cells in B were collected after 0, 2, 4, 6 and 8 hours following addition of β-estradiol/ethanol and total cell extracts were prepared. Immunoblotting was performed using an anti-HA antibody and anti-tubulin antibody. The lane labelled C on the western image on the right contained extracts from β-estradiol treated wild type cells (t = 4 hours) and served as a positive control. D) Aliquots of cells in B were collected after 0, 2, 4, 6 and 8 hours following addition of β-estradiol and total RNA from the cells was prepared. IME1 mRNA levels were quantified by quantitative RT-PCR and normalized with respect to ACT1 mRNA. In the graph, IME1 mRNA levels are expressed relative to IME1 mRNA levels in cells before induction of IME1 expression (t = 0 h).
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Figure 7. Endosulfine mutant is defective in pre-meiotic autophagy but not for autophagy induced by rapamycin treatment.A) Wild type, PCLB2CDC55, igo1Δ igo2Δ and igo1Δ igo2Δ PCLB2CDC55 cells expressing GFP-Atg8 were induced to sporulation. Samples were collected at indicated times and total cell extract was prepared. Western analysis was performed using anti-GFP antibody. B) Wild type, cdc55Δ, igo1Δ igo2Δ and igo1Δ igo2Δ cdc55Δ cells expressing GFP-Atg8 were grown to log phase in YEPD and rapamycin (final concentration 200 ng/ml) was added to the cultures. The cultures were incubated further for 3 hours. Cells were collected at indicated time points, total protein extract was prepared and immunoblot analysis was performed using anti-GFP antibody. C) Wild-type, igo1Δ igo2Δ cells were grown to saturation in YEPD, rapamycin (final concentration 200 ng/µl) was added to the culture and grown for another 24 hours. Sporulation efficiency was measured by counting the total number of monad, dyad and triads/tetrads in the culture (n = 200).
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Figure 8. Regulation of entry into gametogenesis and quiescence by the Rim15-Endosulfine-PP2ACdc55 signalling module.Phosphorylation of endosulfine by Rim15 converts it into an inhibitor of PP2ACdc55 and thus leading to entry into gametogenesis and quiescence. Entry into quiescence is driven by activation of transcription factors Msn2, Msn4 and Gis1. PP2ACdc55 might inhibit a positive regulator of entry into gametogenesis and quiescence. Alternatively, PP2ACdc55 could inhibit entry into quiescence and gametogenesis by dephosphorylating distinct substrates.
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Figure 1. Endosulfines are required for entry into gametogenesis.A) Wild-type, igo1Δ, igo2Δ and igo1Δ igo2Δ cells were incubated for 24 hours on sporulation plates and number of sporulated (includes monad, dyad, Tri-/tetrads) and unsporulated cells were counted using a light microscope. The experiment was repeated 3 times and 200 cells were counted every time for each strain. B) Wild-type and igo1Δ igo2Δ cells were transferred to sporulation medium (SPM) and DNA content was measured by flow cytometry. C) Kinetics of nuclear division of cells in B was measured after staining cells with DAPI (n = 200). D) Analysis of expression of early meiotic proteins in cells described in B. Whole-cell extracts of hourly culture in SPM were subjected to western analysis using anti-PK (to detect Ime1), anti-HA (to detect Rec8) and Cdc28 antibody (loading control). The lane indicated as M contains mitotic extracts and * indicate a non-specific band. E) Conserved Rim15/Greatwall kinase site present in endosulfines from Saccharomyces cerevisiae (S.c), Kluyveromyces lactis (K.l.), Candida glabrata (C.g.), Caenorhabditis elegans (C.e.), Drosophila melanogaster (D.m.), Arabidopsis thaliana (A.t.) and humans (H.s.) is indicated. F) igo1Δ igo2Δ cells and igo1Δ igo2Δ cells containing either pRS303-IGO1-myc8 or pRS303-IGO1-S64A-myc8 or pRS303-IGO1-S64D-myc8 were incubated for 24 hours on sporulation plates and number of sporulated (includes monad, dyad, triads/tetrads) and unsporulated cells were counted using a light microscope. Values are expressed as mean ± s.e.m of 3 independent measurements. *P<0.01 (Student's t-test).
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