MAP kinase Pathways in Yeast

How are signals specified between pathways that share Common components?

MAP = mitogen activated protein kinase

PRP

Ste2/3/4/18

Cdc42/Ste20

Ste11Ste7Fus3

Ste12

Ste5

FGP

?

Cdc42/Ste20

Ste11Ste7Kss1

Ste12/Tec1

Two MAP kinase pathways

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 enzyme E1

High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E1 active site (ATP/AMP)

Ubiquitin conjugating enzymes E2

Ub is transferred to a Cys of 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

UbUb

E1E1

UbUb

E2E2

UbUb

E3E3

UbUb

26s proteosome degradation26s proteosome degradation

TargetTarget

The Ubiquitin PathwayThe Ubiquitin Pathway

UbUbUbUbUbUb

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.