Interaction between red-type Rubisco and the unfoldase ClpX
Transcript of Interaction between red-type Rubisco and the unfoldase ClpX
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Interaction between red-type Rubisco and the unfoldase ClpX
Tihomir Todorov
Laboratory of Prof. Dr. Franz-Ulrich HartlSupervisor Javaid Bhat
Department of Cellular BiochemistryMax-Planck Institute of Biochemistry
19.08.2015
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Project background
Rubisco and the Calvin-Benson-Bassham cycle
RuBP – ribulose-1,5-bisphosphate3PG – 3-phosphoglycerateG3P – glyceraldehyde-3-phosphate
Adapted from Hauser, T., Hartl F. U. et al., (2015), Nature Plants
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Project backgroundRubisco inactivation and reactivationRubisco activase (Rca)
• red-type Rubisco(from Rhodobacter spheroides);
• Rubisco activase – CbbX;
Hauser, T., F. U. Hartl et al., (2015), Nature Plants
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Project background
The unfoldases ClpA and ClpX
ssrA tag
Unfolded substrate
ClpX /
Adapted from Hanson, P. I. and S. W. Whiteheart, (2005), Nature Mol. Cell Biol.
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Project
Hypothesis:
The interaction between ClpX and the inhibited ssrA-tagged Rubisco leads to the reactivation of Rubisco.
Main goal:Insight on the function of Rubisco activase (CbbX).
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Project • Known so far:
activase
Rubisco – RuBP(inhibited)
Rubisco (activated)
RuBP
• To be understood:
ClpX+
Rubisco-ssrA – RuBP(inhibited)
a different interaction
??? Rubisco activation
CbbX and ClpX Same mechanisms
CbbX and ClpXDifferent mechanisms
Adapted from Stotz, M. et al., (2011), Nature
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1. Purification of the required proteins
2. Assays
Project
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Project 1. Purification of the required proteins:
1. Rubisco ssrA-tagged (from R. spheroides)
2. ClpX3. ClpA4. ClpP
5. GFP ssrA-tagged (substrate)
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Project 2. Coupled assay for ATP hydrolysis
ClpX + Rubisco-ssrA–RuBP + ATP (inhibited) ADP + Pi
RuBPReactivated Rubisco
NADH
NAD+
LDH
PEP – phosphoenolpyruvatePK – phosphokinaseLDH – lactate dehydrogenase
PK + PEP
ATP + pyruvate
lactic acid
Monitored at
A340 Adapted from Barta, C. et al. (2011) Rubisco Activase Activity Assays. Photosyth Res Prot, 375-382
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by 2050More than 25% in the global food production
Evans, J. & von Caemmerer, S. Enhancing photosynthesis. Plant Physiol. 155, 19 (2011)
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Acknowledgements
Prof. Dr. Franz-Ulrich HartlDr. Manajit Hayer-HartlJavaid BhatNadine WischnewskiAnastasia Jungclaus
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ssrA tag:
AANDENYALAA
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Rubisco inhibitors:
RuBP – ribulose-1,5-bisphosphateKABP – 3-ketoarabinitol-1,5-bisphosphateXuBP – xilulose-1,5-bisphophatePDBP – D-glycero-2,3-pentodiulose-1,5-bisphosphateCTBP – 2-carboxytetritol-1,4-bisphosphate
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Structure of red-type Rubisco
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Red-type Rubisco
Green-type Rubisco
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Rubisco activation
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CO2 fixation