Prof. Dr. Michael Schroda

Using the unicellular green alga Chlamydomonas reinhardtii as model organism, we are working on four main projects:

• Functional investigation of the chloroplast HSP70/HSP90 chaperones
• Investigation of the mechanisms by which the HSP70A promoter activates transgene expression
• Dissection of the sterss response in Chlamydomonas
• GoForsys

1. Functional investigation of the chloroplast HSP70/HSP90 chaperones

Rationale

Chaperones are a specialized class of proteins, whose most well-known function is to help other proteins to assume and maintain the native folding state. In addition, chaperones participate in numerous other activities like the translocation of proteins across biomembranes, protein complex assembly/disassembly, maturation of signal transduction components, tagging of unfoldable proteins for degradation, etc. While chaperones have been extensively studied in bacteria, mitochondria, the ER, and the eukaryotic cytosol, comparably little is known about their activities in chloroplasts. This is surprising, since chloroplasts harbour a unique compartiment which is the ultimate energy source for almost all life on earth - the thylakoid membranes. Moreover, in light of the growing importance of the chloroplast as the compartment of choice for the expression of recombinant proteins an understanding of its protein folding machinery is indispensable.

Aim

To biochemically characterize the chloroplast chaperone networks and to elucidate their roles in chloroplast biogenesis and maintenance of chloroplast functions.

Current State

We have identified and biochemically characterized several components of the chloroplast HSP70 and HSP90 chaperone systems in Chlamydomonas. Moreover, we have gained insights into the regulation of HSP70B’s chaperone activity and identified one of its substrates. The current state of knowledge is summarized below:
Click the image to see a larger version with description.

Currently addressed questions and approaches

• Which are the substrates of the chloroplast HSP70 and HSP90 systems?
  → We apply QUICK-X, which by a combination of RNAi, stable isotope labelling, immunoprecipitation, and quantitative mass spectrometry allows for the identification of protein-protein interactions at high sensitivity.
• How does HEP2 activate HSP70B after import into the chloroplast?
  → We generate homology models of HSP70B and HEP2 and simulate their interactions by the RosettaDock program. Moreover, we crosslink recombinant HSP70B and HEP2 and identify dipeptides by mass spectrometry. Deduced interaction surfaces are verified by site-directed mutagenesis.
• Why do the HSP90/HSP70 systems interact with VIPP1?
  → We investigate the function of VIPP1 in the chloroplast by analysing VIPP1-RNAi strains at the levels of physiology and proteome composition using quantitative mass spectrometry. Moreover, we seek for more VIPP1 interacting proteins with QUICK-X.
• Is HSP70B redox-regulated?
  → We investigate the chaperone activity of HSP70B in vitro under certain redox states. Moreover, we mutate the redox-active cysteines in HSP70B and analyse effects of redox stress in vitro and in vivo