Yamada A , Duffy B , Perry JA , Kornbluth S .

R01GM067225 NIGMS NIH HHS , R01 GM067225 NIGMS NIH HHS

	Figure 1. Amino acid sequence alignment of Xenopus, human and yeast Hsl7 proteins. The amino acid sequences of Hsl7 orthologs were aligned using the ClustalW alignment parameters in the program MacVector. Dark shading indicates identities and light shading indicates similarities.
	Figure 2. Excess xHsl7 accelerates entry into mitosis in a nuclear-dependent manner. (A) GST-yeast Hsl7 (lane1) or GST (lane 2) were incubated in Xenopus egg extracts, retrieved on glutathione Sepharose, and immunoblotted with anti-Wee1 antibodies. (B) xHsl7-encoding mRNA or buffer was incubated in cycling extracts with sperm nuclei (5,000/μl) and an ATP-regenerating system. Aliquots were stored at the indicated times and assayed for their ability to phosphorylate histone H1 in the presence of [32P]ATP. Phosphorylated histone was resolved by SDS-PAGE, subjected to autoradiography, and quantified by phosphoimager with the zero time point normalized to 1. Square, buffer; circle, FLAG-xHsl7 mRNA addition. Arrows indicate the time of the nuclear envelope break down and the chromosome condensation as monitored by microscope. (C) Same assay as panel B except that sperm nuclei were absent. (D) In vitro–translated 35S radiolabeled xHsl7 and nucleoplasmin (as an internal control) were added to cycling extracts with or without nuclei present. Samples were withdrawn at the indicated times, resolved by SDS-PAGE, and subjected to autoradiography to monitor xHsl7 stability. Mitosis was observed at 90 min by fluorescence microscopy of extracts containing Hoechst-stained nuclei.
	Figure 3. Xenopus Hsl7 interacts with Wee1 in Xenopus egg extracts. (A) HeLa cell lysate (10 μl), Xenopus egg extracts (2 μl), or GST-xHsl7 were resolved by SDS-PAGE and immunoblotted with anti-JBP1. (B) Immunoprecipitates formed using affinity-purified anti-Wee1 or control IgG were immunoblotted with anti-JBP1. (C) GST or GST-xHsl7 coupled to glutathione Sepharose was incubated in Xenopus egg extract and immunoblotted with anti-xWee1.
	Figure 4. Blocking xHls7 function delays mitotic entry. (A) Cycling extracts were depleted with antibodies directed against xHsl7 (square) or with control mouse IgG (circle). Depleted extracts were incubated at room temperature with sperm chromatin and an ATP-regenerating system, and cell cycle progression was monitored by measuring Histone H1-directed kinase activity, beginning at 60 min when both extracts were still in interphase, as detected by fluorescence microscopy of Hoechst-stained nuclei. (B) Control IgG (closed circle), anti-Hsl7 (closed square), or anti-Hsl7 supplemented with recombinant xHsl7 (open triangle) was incubated in 100 μl of cycling extract supplemented with sperm nuclei and ATP-regenerating system. Experimental analysis was identical to Fig. 2 B. (C) Hoechst staining of DNA was monitored by fluorescence microscopy to visualize nuclear envelope breakdown and chromatin condensation.
	Figure 5. Excess xHsl7 restores inhibition of oocyte maturation induced by Wee1 injection and xHsl7 does not affect Wee1 kinase activity. (A) 50 stage VI oocytes were injected with 40 ng of mRNAs encoding β-globin (open square), FLAG-xHsl7 (open circle), HA-Wee1 (closed square), and 40 ng each of FLAG-xHsl7 and HA-Wee1 mRNAs together (closed circle). After a 12-h incubation, they were treated with progesterone and scored for the percentage of GVBD. (B) Ultra S Xenopus egg extract (purified cytosol) was either mock depleted, depleted of xHsl7, or supplemented with recombinant xHsl7. ATP-regenerating system was then added along with sodium vanadate to inhibit dephosphorylation of Cdc2. Recombinant human cyclin B1 was then added and the reaction was allowed to proceed for 10 min. xWee1 activity was determined by assaying the phosphorylation level of Cdc2 by immunoblotting with anti-phospo Cdc2.
	Figure 6. Hsl7 promotes intranuclear Wee1 degradation. (A) Xenopus stage VI oocytes were injected with 40 ng of β-globin or FLAG-xHsl7 mRNAs and incubated in the presence of 50 μM roscovitine. 12 h later, they were injected again with 35S-labeled Wee1 protein. After the second injection, they were treated with progesterone (1 μM) and roscovitine in modified Barth's + Ca buffer. At the indicated times after treatment, five oocytes were manually dissected into nuclear (left) and cytoplasmic (right) fractions, and analyzed by SDS-PAGE and autoradiography. The graph represents a quantitation of the data above showing the fraction of Wee1 remaining in nuclei or cytosol. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. The bars to the left of the panel denote the mobility shift of Wee1. (B) 40 ng mRNAs of FLAG-xHsl7 (circle) and FLAG-xHsl7 metyltransferase mutant (triangle) were injected into oocytes. The Wee1 degradation assay was performed as described in panel A. The graph shows the fraction of Wee1 remaining in nuclei. (C) Oocytes were injected as in panel A, but after the second injection, they were treated with leptomycin B (200 nM) for 2 h. Progesterone (1 μM) was then added to induce oocyte maturation. At the indicated times after progesterone treatment, five oocytes were manually fractionated and analyzed by SDS-PAGE followed by autoradiography. The graph shows the fraction of Wee1 remaining in nuclei. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. (D) The experiment in Fig. 5 A was repeated as described except that MG132 was injected into the oocytes along with the indicated mRNAs. Open square, β-globin; open circle, FLAG-xHsl7; closed square, HA-Wee1 mRNA injection; closed circle, FLAG-xHsl7 and HA-Wee1 mRNAs coinjection. (E) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2 and 3) were incubated in cycling extracts in the presence (lanes 1 and 3) or absence (lane 2) of sperm chromatin. After 60 min, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (F) Interphase (lanes 1 and 3) or mitotic (lane 2) egg extracts were incubated in the presence of sperm chromatin for 60 min, and immunoprecipitated with anti-Wee1 antibody (lanes 1 and 2) or control antibody (lane 3) bound to protein A–Sepharose beads. Immunoprecipitates were then blotted with anti-Hsl7 antibodies.
	Figure 7. Excess Hsl7 overrides the DNA replication checkpoint. (A) Xenopus Hsl7 or β-globin mRNA was incubated in cycling extracts in the presence of aphidicolin (200 μg/ml). Cell cycle progression was monitored by assessing histone H1 kinase activity. Square, β-globin mRNA; circle, FLAG-xHsl7 mRNA. An arrow indicates the time of the nuclear envelope break down and the chromosome condensation as monitored by a microscope. (B) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2,3, 4) were incubated in cycling extracts containing no sperm (lane 2) or sperm chromatin DNA (lanes 1, 3, and 4) in the absence (lanes 1–3) or presence (lane 4) of aphidicolin (200 μg/ml). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (C) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2–4) were incubated in cycling extracts containing sperm chromatin DNA in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of aphidicolin (200 μg/ml) and inactive Plx1 (lane 3) or constitutively active Plx1 (T201D; lane 4). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting.

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