Project Leader

Alexander Graf
Project Leader
Phone: +49 331 567 8113

Department Stitt

Plant Proteomics

The group of Dr Alexander Graf studies how the formation of protein complexes and posttranslational modification of proteins contribute to the regulation of metabolic pathways in the model plant Arabidopsis thaliana. High throughput cutting-edge mass spectrometric analyses of the plant proteome combined with bioinformatics data-analysis play a central role in our projects. However, we also employ classic genetic and biochemical methods to study gene functions in focused approaches.

Protein complexes

All cellular functions in living organisms are carried out by proteins. It is well known that most proteins are not active alone but interact with other proteins to form complexes. The interaction of proteins and the formation of protein complexes have been studied for many years with a range of in vitro and in vivo methods. Most of these methods only allowed the identification of a limited number of proteins. These include yeast-two-hybrid interaction studies, co-immunoprecipitations and bi-fluorescence complementation assays. Commonly used 2-D gels combined with mass spectrometric analyses can also not deliver more than a few hundred proteins within one experiment. Moreover, none of the methods mentioned above allow reliable quantification of protein abundances.

We use native gel electrophoresis and gel filtration to fractionate protein complexes from plant extracts. We combine these classic methods with high throughput mass spectrometric analysis, which allow the detection and quantification of several thousand proteins in one experiment. In the first phase of this project, we will establish a map of protein complexes in Arabidopsis. The confirmation of previously published protein-protein interactions will serve as a backbone to which predictions of new interactions will be added. Experiments to confirm predicted components of protein complexes will include tap-tag immunoprecipitations and functional relationships will be unravelled using classic genetic and biochemical methods. In the second phase, the established map will be used to quantitatively study changes in protein complex abundances and complex composition in different environmental conditions and genetic backgrounds. These experiments will enable us to draw conclusions about the role of protein complex formation in the regulation of metabolic pathways in Arabidopsis.

Protein SUMOylation

Posttranslational modification of proteins by Small Ubiquitin-like modifiers (SUMOs) is closely related to protein ubiquitination. While ubiquitination primarily targets proteins for degradation via the proteasome, SUMOylation has been shown to affect protein function and localisation. The SUMO peptide is transferred to lysine residues of the target protein by SUMO ligases. The modification can be removed by SUMO peptidases.

Current methods to study protein SUMOylation rely on enrichment strategies like immunoprecipitaion using SUMO antibodies or employ mutant plants complemented with a modified SUMO peptide. As a first part of the SUMO project, we establish a method allowing the proteome wide detection and quantification of SUMOylated proteins by mass spectrometry. To unravel the role of SUMOylation in the regulation of plant metabolism, we will employ this method to quantitatively study SUMOylated protein in different environmental conditions.

The second part of this project is focused specifically on the regulation of enzyme activities in the central carbohydrate metabolism by protein SUMOylation. Detailed analysis of mutants with defects in the SUMOylation pathway will be performed on the transcript and metabolite level. We want to answer the question whether SUMOylation allows plants to rapidly adjust enzyme activities to changes in environmental conditions.

 
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