1. [Vierstra, 2003 TIPS]

2. [Vierstra, 2003 TIPS]

3. Ubiquitin/26S proteasome pathway
Simplified
Ubiquitination
Proteolysis
+ ATP Ub E1 E2 E3 Ub Ub Ub Ub
Target
Target
+ ATP
26S proteasome

4. Loss of 26S proteasome functionWT
rpn10-1
Smalle et al., 2003
Loss of proteasome function in the rpn10-1 mutant, leads to growth inhibition and the accumulation of polyubiquitinated target proteins.

5. Diversity in Ubiquitination Machinery is largely provided by the many E3s
Single E1
Few E2’s
Many E3’s

6. E3 structure/function Ub E2 Target protein E3 (Ubiquitin ligase)
Target binding
E2 binding Ub E2
Target protein
E3 (Ubiquitin ligase)
Target binding
E2 binding

7. E3 structure/function Ub Ub Ub Ub Ub Target protein Ub Ub Ub Ub
26S proteasome Ub Ub Ub Ub
Target protein E3
Target binding
E2 binding

8. Number of E3s per genome Saccharomyces cereviseae 68
Caenorhabditis elegans
Drosophila melanogaster
Homo sapiens
Arabidopsis thaliana 68
657
189
527
1156

9. Advantages of proteolysis control in signal transduction
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.

10. Why more E3s in plants?
* 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.
From Kepinski and Leyser, 2003

11. Proteolysis control of signaling
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).

12. 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.

13. 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.

14. * Signal (variable) DNA RNA Response repressor Response repressor E3 R
Control of gene expression via conditional proteolysis
EXAMPLE 1:
Signal (variable) *
DNA
RNA
Response repressor
Response repressor
Constitutive expression E3 R e s p n r o
Response (variable)

15. * Signal (variable) DNA RNA Response activator Response activator R e
Control of gene expression via conditional proteolysis
EXAMPLE 2:
Signal (variable) *
DNA
RNA
Response activator
Response activator
Constitutive expression R e s p n a c t i v r o E3
Response (variable)

16. ABA response
(Vierstra, 2009)

17. Signal (variable) E3 R e s p n a c t i v r o DNA RNA
Control of gene expression via conditional proteolysis
EXAMPLE 3:
Signal (variable) E3 R e s p n a c t i v r o
DNA
RNA
Response activator
Constitutive expression
Response (variable)

18. Light responses (variable)
Control of gene expression via conditional proteolysis
EXAMPLE 3: Photomorphogenesis
Light (variable)
COP1 R e s p n a c t i v r o
DNA
RNA
HY5
Constitutive expression
Light responses (variable)

19. COP1 acts as an E3 to target HY5 for degradation
RING
Coil
WD-40 repeats
COP1
E2* Ub
bZIP
HY5
HY5 Ub Ub E2 Ub Ub
Degradation via
26S Proteasome
Degradation by the 26S proteasome Ub E1 Ub
(Osterlund et al.,2000)

20. Photomorphogenesis LIGHT Light intensity COP1 HY5 LIGHT RESPONSES HY5
(Osterlund et al., 2000)

21. Signal (variable) E3 R e s p n r o DNA RNA Response repressor
Control of gene expression via conditional proteolysis
EXAMPLE 4:
Signal (variable) E3 R e s p n r o
DNA
RNA
Response repressor
Constitutive expression
Response (variable)

22. Auxin Response (variable)
Control of gene expression via conditional proteolysis
EXAMPLE 4: Auxin response pathway
Auxin (variable)
TIR1 R e s p n r o
DNA
RNA
AUX/IAA factors
Constitutive expression
Auxin Response (variable)

23. Auxin response
(Vierstra, 2009)

24. Jasmonate response
(Vierstra, 2009)

25. Summary: important to remember
How does a target protein become polyubiquitinated through the sequential action of E1, E2 and E3 enzymes?
26S Proteasome: structure/function. How does the proteasome detect and then degrade target proteins?
Where in the cell does the Ubiquitin/26S Proteasome pathway act?
ATP requiring steps in the pathway? Energy is needed to establish specific proteolysis (as opposite to non-specific).
Predict the effects of loss of function of different components of the pathway (proteasome --- pleiotropic; E3 --- highly specific phenotype).
Why proteolysis control of signal transduction (what are the advantages)?
Possible mechanisms of conditional protein degradation to control signal/response ratios (see examples 1-4).

26. 2011

27. 2009

28. 2008