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[Vierstra, 2003 TIPS]
[Vierstra, 2003 TIPS]
Ubiquitin/26S proteasome pathway
UbUb
+ ATP
E1
E3
E2
Target
UbUbUbUb
UbUbUbUb
Target
26S proteasome
Ubiquitination Proteolysis+ ATP
Simplified
WT rpn10-1
Loss of 26S proteasome function
Smalle et al., 2003
Loss of proteasome function in the rpn10-1 mutant, leads to growth inhibition and the accumulation of polyubiquitinated target proteins.
Diversity in Ubiquitination Machinery is largely provided by the many E3s
Single E1
Few E2’s
Many E3’s
E3 structure/function
E2 bindingTarget binding
UbE2
Target protein
E2 bindingTarget binding
Target proteinUb
E2
E3 (Ubiquitin ligase)
E3 (Ubiquitin ligase)
E3 structure/function
Ub
E2 bindingTarget binding
Target protein
E3
UbUb
Ub
Ub
Target protein
Ub Ub
Ub
26S proteasome
UbUb
UbUb
Number of E3s per genome
68
657
189
527
1156
Saccharomyces cereviseae
Caenorhabditis elegans
Drosophila melanogaster
Homo sapiens
Arabidopsis thaliana
1) Fast response to a change in signal intensity:
direct control of protein activities in contrast to transcriptional regulation that involves transcription, transcript processing and translation steps before protein abundance is increased.
2) Proteolysis control can rapidly increase as well as
decrease a proteins activity (only an increase is possible with transcriptional regulation).
3) Accurate reflection of signal intensity in response
output: secundary modifications such as phosporylation/dephosphorylation can also directly change a proteins activity. However since such controls tend to be leaky, i.e. are the result of modification/demodification equilibria, their outcome depends on the initial abundance of the target protein.
Advantages of proteolysis control in signal transduction
Why more E3s in plants?
From Kepinski and Leyser, 2003
* More E3s means more proteolysis control of signaling.* Energetically wasteful?* Animals can side-step adverse environmental conditions.•The sessile plant must endure.•Plants need to be more sensitive to environmental changes.* Proteolysis control of signaling allows for quick responses to changes in signal intensities (changes in environmental conditions).* Proteolysis control also allows for an accurate response-strength to signal-intensity ratio.* Allows for a constant state of readiness.* Plants are less energy-limited.
Signal transduction leads to destabilization of a repressor of the response or stabilization of a response activator.
This is accomplished via secundary modification (phosphorylation or dephosphorylation) of the target protein that leads to or prevents its detection by a Ubiquitin ligase (E3). Alternatively, signaling directly controls E3 affinity for the target protein.
Controlling the activity of a protein via its degradation rate allows for faster and more accurate responses to changing concentrations/intensities of the signal (changing environment).
Proteolysis control of signaling
The Ub/26SP pathway and signaling
Describe two mechanisms that can be used to transform a signal into a response via the regulated degradation of a repressor of this response. Show how increased signal intensity leads to an increased response output.
The Ub/26SP pathway and signaling
Describe two mechanisms that can be used to transform a signal into a response via the regulated degradation of an activator of this response. Show how increased signal intensity leads to an increased response output.
Response repressor
*
Signal (variable)
Response (variable)
DNA RNA Response repressorConstitutive expression
Re
spn
se
ro
os
seer
r p
E3
Control of gene expression via conditional proteolysisEXAMPLE 1:
Response activator
*Signal (variable)
Response (variable)
DNA RNA Response activatorConstitutive expression
Re
spn
se
a
cti vro
o
E3
Control of gene expression via conditional proteolysisEXAMPLE 2:
ABA response
(Vierstra, 2009)
Response activator
Signal (variable)
Response (variable)
DNA RNA
Re
spn
se
a
cti vro
oConstitutive expression
E3
Control of gene expression via conditional proteolysisEXAMPLE 3:
HY5
Light (variable)
Light responses (variable)
DNA RNA
Re
spn
se
a
cti vro
oConstitutive expression
COP1
Control of gene expression via conditional proteolysisEXAMPLE 3: Photomorphogenesis
COP1 acts as an E3 to target HY5 for degradation
RING Coil WD-40 repeats
E2 Ub
E1 Ub
E2*bZIP
Degradation via 26S Proteasome
COP1
HY5Ub
Ub
(Osterlund et al.,2000)
Ub
Ub
Ub
COP1
HY5
Degradation by the 26S proteasome
Photomorphogenesis
Light intensity
(Osterlund et al., 2000)
HY5
HY5
LIGHT
COP1
LIGHT RESPONSES
Response repressor
Response (variable)
DNA RNA Constitutive expression
Signal (variable)
E3
Re
spn
se
ro
os
seer
r p
Control of gene expression via conditional proteolysisEXAMPLE 4:
AUX/IAA factors
Auxin Response (variable)
DNA RNA Constitutive expression
Auxin (variable)
TIR1
Re
spn
se
ro
os
seer
r p
Control of gene expression via conditional proteolysisEXAMPLE 4: Auxin response pathway
Auxin response
(Vierstra, 2009)
Jasmonate response
(Vierstra, 2009)
1) How does a target protein become polyubiquitinated through the sequential action of E1, E2 and E3 enzymes?
2) 26S Proteasome: structure/function. How does the proteasome detect and then degrade target proteins?
3) Where in the cell does the Ubiquitin/26S Proteasome pathway act?
4) ATP requiring steps in the pathway? Energy is needed to establish specific proteolysis (as opposite to non-specific).
5) Predict the effects of loss of function of different components of the pathway (proteasome --- pleiotropic; E3 --- highly specific phenotype).
6) Why proteolysis control of signal transduction (what are the advantages)?
7) Possible mechanisms of conditional protein degradation to control signal/response ratios (see examples 1-4).
Summary: important to remember
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