interaction between red-type rubisco and the unfoldase clpx
TRANSCRIPT
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
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
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
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.
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).
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
1. Purification of the required proteins
2. Assays
Project
Project 1. Purification of the required proteins:
1. Rubisco ssrA-tagged (from R. spheroides)
2. ClpX3. ClpA4. ClpP
5. GFP ssrA-tagged (substrate)
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
by 2050More than 25% in the global food production
Evans, J. & von Caemmerer, S. Enhancing photosynthesis. Plant Physiol. 155, 19 (2011)
Acknowledgements
Prof. Dr. Franz-Ulrich HartlDr. Manajit Hayer-HartlJavaid BhatNadine WischnewskiAnastasia Jungclaus
ssrA tag:
AANDENYALAA
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
Structure of red-type Rubisco
Red-type Rubisco
Green-type Rubisco
Rubisco activation
CO2 fixation