1. MAP kinase Pathways in Yeast How are signals specified between pathways that share Common components? MAP = mitogen activated protein kinase

2. PRP Ste2/3/4/18 Cdc42/Ste20 Ste11 Ste7 Fus3 Ste12 Ste5 FGP ? Cdc42/Ste20 Ste11 Ste7 Kss1 Ste12/Tec1 Two MAP kinase pathways

4. UBIQUITIN ● 76 Amino Acid polypeptide ● Highly conserved in evolution: 3 Amino acid differences between yeast and human homologues

5. Degradation of a Protein Via the Ubiquitin-Proteasome Involves Two Successive Steps ● 1. Covalent attachment of multiple ubiquitin molecules to a protein substrate. ● 2. Degradation of the tagged protein by the 26s proteasome. (ubiquitin is recycled)

6. UBIQUITINATION In general, multiple ubiquitin units are arranged in polyubiquitin chains linked via Lys 48 of ubiquitin, targeting the protein for degradation

7. Ubiquitin Conjugation: A 3 Step Mechanism Ubiquitin (Ub) activating enzyme E 1 High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E 1 active site (ATP/AMP) Ubiquitin conjugating enzymes E 2 Ub is transferred to a Cys of E 2 forming a new thiol ester Ubiquitin ligase E 3 Ub forms isopeptide bond between C-terminal Gly of Ub and ε-amino group of Lys on a target protein

8. Ub E1 Ub E2 Ub E3 Ub 26s proteosome degradation Target The Ubiquitin Pathway Ub Ub Ub

9. Classes of E3 ligases ● N-end Rule E3 ● Skp/Cullin/F-box (SCF) ● Anaphase Promoting Complex ● HECT family ● RING finger domain

10. Phosphorylation of I   leads to ubiquitination and degradation of the inhibitor and activation of the transcription factor.

11. ATP consuming process

12. THE PROTEASOME

13. Small Ubiquitin Related Modifier (SUMO) ● SUMO does not have the Lys-48 found in ubiquitin ● SUMO does not make multi-chain forms ● SUMO-1,2,3 are the mammalian forms ● SUMO-1, 101 amino acids, C-terminal Gly, 18% identical to ubiquitin.

14. SUMO

15. MASS-SPEC Analysis Allows determination of peptide sequences and modified peptides (by P~) Mass spectrometers separate gas-phase ions according to their mass to charge ratios The analyzer uses a combination of magnetic and electric fields to move ions from the regions that they are produced to the detector.