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MAP kinase Pathways in Yeast How are signals specified between pathways that share Common components? MAP = mitogen activated protein kinase Two MAP kinase pathways PRP FGP Ste2/3/4/18 ? Cdc42/Ste20 Cdc42/Ste20 Ste11 Ste7 Fus3 Ste11 Ste7 Kss1 Ste12 Ste5 Ste12/Tec1 Two MAP kinase pathways UBIQUITIN ● ● 76 Amino Acid polypeptide Highly conserved in evolution: 3 Amino acid differences between yeast and human homologues 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) UBIQUITINATION In general, multiple ubiquitin units are arranged in polyubiquitin chains linked via Lys48 of ubiquitin, targeting the protein for degradation Ubiquitin Conjugation: A 3 Step Mechanism Ubiquitin (Ub) activating High energy thiol ester is enzyme E1 formed between C-terminal Gly of ubiqutin and a Cys in the E1 active site (ATP/AMP) Ubiquitin conjugating Ub is transferred to a Cys of E2 enzymes E2 forming a new thiol ester Ubiquitin ligase E3 Ub forms isopeptide bond between C-terminal Gly of Ub and ε-amino group of Lys on a target protein The Ubiquitin Pathway Ub E1 Ub Ub E2 E3 Target Ub Ub Ub Ub 26s proteosome degradation Classes of E3 ligases ● N-end Rule E3 ● Skp/Cullin/F-box (SCF) ● Anaphase Promoting Complex ● HECT family ● RING finger domain Phosphorylation of I leads to ubiquitination and degradation of the inhibitor and activation of the transcription factor. ATP consuming process THE PROTEASOME 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. SUMO 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.