1971j. of Exp. Zoology

Criteria for CSF

1) It should appear in the oocyte cytoplasm after Progesterone treatment

2) It should cause nuclei to arrest in metaphase

3) It should be maintained until the oocyte is activated

4) It should become inactivated during fertilization or egg activation

Assays used to investigate CSFA and B are gain of function experiments. C is a loss of function assay.

Nature 1989

Metabolic label with [35S] Methionine

Metabolic labeled with [32P] ATP

In the three cases, the experiments showed autoradiographies after IP with anti Mos

Synthesis and Modification of Mos during oocyte maturation

35S labeling followed by IP with anti Mos

Slight shift in MW

Antigenic peptide:

Criteria 1) It should appear in the oocyte cytoplasm after Progesterone treatment

Selective proteolysis of Mos upon egg activation

In vitro matured oocytes pre-labelled with [35S]Methionine were washed and chased for one hour without activation (none/chase), or after activation by either pricking with a glass nnedle (Prick/chase), or treatment with Ca2+ ionophore (A23187/chase). B. Time curve of Mos degradation.

Criteria 4) It should become inactivated during fertilization or egg activation

Criteria for CSF

1) It should appear in the oocyte cytoplasm after Progesterone treatment

2) It should cause nuclei to arrest in metaphase

3) It should be maintained until the oocyte is activated

4) It should become inactivated during fertilization or egg activation

1989

Absence of Mos protein in Xenopus fertilized eggs

Autoradiography Western blot

Oocytes are pre labeled with 35S Methionine and then immunoprecipitated with anti Mos. Immunoprecipitates are then analyzed by PAGE and Mos visualized either by autoradiography or by Western blot using anti Mos.

Labeling is done after fertilization for 2 hours. Why is the WB an important experiment?

3) It should be maintained until the oocyte is activated

4) It should become inactivated during fertilization or egg activation

Morphology of embryos and chromosomes as well as MPF activity in mos RNA-arrested embryos

In these experiments mos RNA was injected in one of the cells of a two-cell embryo. a. Morphology. b. Chromosome staining. C. MPF activity from cytosol of mos-arrested embryos in immature oocytes.

CN-Mos: capped normal Mos

Criteria 2) It should cause nuclei to arrest in metaphase

MPF activity

Arrested embryo with capped Mos mRNA

Cleavage arrest induced by injected RNA

CN-mos: Capped mos mRNACN-mos + sense: same + sense mRNAUN-mos: Uncapped mos mRNACT-mos: Capped truncated (minus kinase domain) mos RNAUT-mos: Uncapped truncated mos RNATMV: tobacco mosaic virus total mRNA (Amersham)Globin: b=globin mRNA tRNA: Yeast tRNA (Sigma)Xenopus oocyte: total mRNA from Immature Xenopus oocytes

Criteria 2) It should cause nuclei to arrest in metaphase

Effect of Mos-specific antibodies on CSF activity

B-Mos: Polyclonal antibody against Mos. It is able to block Mos kinase activity in vitro

5S: Monoclonal antibody against Mos. It does not block Mos kinase activity.

C232: Another monoclonal antibody against Mos.Two types of experiments: 1) Neutralization with specific antibodies. 2) Immunodepletion with specific antibodies

In all these cases CSF extracts were obtained from matured-unfertilized eggs. The treatment with the different antibodies was conducted before microinjection in 2-cell embryos.

Additional criteria: Loss of function experiment

MPF and CSF levels during oocyte maturation and fertilization

Mos has the same activity than Raf; it is a MAP kinase kinase kinase

MEK1 is a Map kinase kinase

P90Rsk is another kinase that mediates the CSF action

Avram Hershko

"for the discovery of ubiquitin-mediated protein degradation"

The Nobel Prize in Chemistry 2004

Aaron Ciechanover Irwin Rose

A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes

Aharon Ciechanover, Yaacov Hod and Avram Hershko1 Technion-Israel Institute of Technology, School of Medicine, Haifa, Israel

Received 8 March 1978.  Available online 02 December 2004.

AbstractThe degradation of denatured globin in reticulocyte lysates is markedly stimulated by ATP. This system has now been resolved into two components, designated fractions I and II, in the order of their elution from DEAE-cellulose. Fraction II has a neutral protease activity but is stimulated only slightly by ATP, whereas fraction I has no proteolytic activity but restores ATP-dependent proteolysis when combined with fraction II. The active principle of fraction I is remarkably heat-stable, but it is non-dialysable, precipitable with ammonium sulfate and it is destroyed by treatment with proteolytic enzymes. In gel filtration on Sephadex-G-75, it behaves as a single component with a molecular weight of approximately 9,000.

Biochem Biophys Res Commun. 1978 Apr 28;81(4):1100-5              A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.Ciechanover A, Hod Y, Hershko A.

To examine the possible association of the heat stable polypeptide with other cellular proteins and the role of the ATP in this process, 125I-APF-1 was incubated with fraction II in the presence of ATP, and was analyzed on columns of Sephadex G-75. On such columns the low MW polypeptide is retained while most of the proteins of fraction II are excluded and eluted at the void volume (V0).

1. - ATP

2-6. + ATPDifferent conditions

JBC 1983

JBC 1981

JBC 1983

Current model of Ubiquitin-induced proteasome-mediated degradation

FERTILIZATION1. Fusion of the sperm and egg plasma membranes.

2. Increase in Calcium (Ca2+) inside the egg.

3. Cortical granule exocytosis.

4. Block to Polyspermy. A) Plasma membrane block to polyspermy.B) Zona block to polyspermy

5. Cyclin Destruction and inactivation of cyclin-dependent kinase (cdk). 6. Sperm Chromosomes

decondensation.

7. Male and Female Pronuclear formation.

8. Syngamy and first mitosis.

Tim Hunt group instead of degrading all the mRNA, they specifically target cyclin B using an antisense approach

Summary of Murray and Kirshner paper

Nature 1991

Two approaches to analyze the sequence important for cyclin degradation: 1) cyclin deletion mutants; 2) addition of sequence to another protein. In this case Protein A.

Protein A-cyclin fusion

Cyclin deletion mutants

Autoradiography 125I proteins are added to Xenopus mitotic or interphase extracts. Degradation is evaluated after different time periods.

Kinetic analysis of degradation of 13-91prA. A) extracts; B) Immunoprecipitated with Ig Sepharose

125I ubiquitin form high Mr complexes with cyclin-Protein A fusion proteins. The autoradiography is developed after immunoprecipitation of Protein A with Ig-Sepaharose.

13-91cyclin-protein A fusion is degraded and it is ubiquitinated. However, 13-91prA-R42C mutant is not degraded and not ubiquitinated.