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