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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
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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
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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
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N-end Rule E3
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Skp/Cullin/F-box (SCF)
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Anaphase Promoting Complex
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HECT family
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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)
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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.