[Vierstra, 2003 TIPS]. Ubiquitin/26S proteasome pathway Ub + ATP E1 E3 E2 Target Ub Target 26S proteasome UbiquitinationProteolysis + ATP Simplified.

[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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