Periodic Reporting for period 1 - GolgiBiosSys (Identification and characterisation of plant Golgi biosynthetic multiprotein systems)
Berichtszeitraum: 2016-05-01 bis 2018-04-30
I use Arabidopsis thaliana as a model organism with the main goal of understanding the proteomic organization of the Golgi apparatus. Probably the most significant functional feature of the plant Golgi apparatus is synthesis of extracellular polysaccharides. These polymers account for at least a third of all renewable organic carbon on the planet and are not only major components of many ecosystems, but also components of our food, fuel, building material, paper and fiber, as well as targets in the search for renewable energy. In terms of its biosynthetic aspects, previous studies have identified enzymes involved in the synthesis of diverse polysaccharides, with evidence that these enzymes are organized in protein complexes as molecular machineries. The organization, stoichiometry and composition of these protein complexes impact their properties as well as the efficiency of their function.
The overall research objective of this project was the application of state of the art quantitative mass spectrometry technologies for the proteomic measurements of plant Golgi protein complexes in order to understand the details of its biosynthetic machineries. More precisely, the first research objective of the proposal aims at investigating the membrane protein interactions in the plant Golgi. The second objective of the proposal consists of identifying specific interacting partners by affinity purification mass spectrometry for partially characterized Golgi membrane protein complexes, such as the cellulose synthase complex. In addition, the training objective of the project is the acquisition of detailed knowledge on quantitative mass spectrometry tools, such as the SWATH-MS technique and related data analysis tools. I also obtained extensive didactic experience by Master student supervision as well as teaching opportunities within the ETH Bachelor student block courses and the international DIA/SWATH course. I gained ample experience in scientific communication, by involvement in organization of scientific meetings, session and outreach activities.
I carried our research on the establishment of a comprehensive tools in order to establish the objectives of the proposal, namely I focused on the generation of a pan-Arabidopsis spectral library that contains spectrum query parameters in order to analyze the mass spectrometry data obtained from SWATH acquisition. The generation of such large scale library required gathering sizable data from different organs and tissues, as well as different growth conditions. In summary, I collected data from 496 mass spectrometry measurements and compiled a repository that covers 13.919 unique proteins which accounts for 50.6% of the total Arabidopsis proteome. This resource will contribute significantly to the simplified and reproducible analysis of Arabidopsis proteome samples across studies and laboratories. The results from this task are currently being prepared for publication and the repository data will be made available as an open resource. This repository is of crucial relevance to the further study on the protein interaction landscape, which I conducted by using a technique that allows high-throughput assignment of protein interactions. The methodological design is based on separation of protein complexes in conjunction with SWATH MS. The separation of intact complexes was achieved by Size Exclusion Chromatography (SEC).
Additionally, I obtained plant lines for the study of specific Golgi membrane protein complexes, namely the cellulose synthase and the xylan synthase complexes. The work on identifying these biosynthetic machineries was carried within a supervision of a Master student and led to the discovery of novel interacting partners involved in the assembly of the cellulose synthase machinery which provides details in the knowledge on plant cell wall synthesis. Additionally I performed affinity purification of another Golgi-localized complex: the xylan synthesis complex and the obtained data support the idea of distinct primary and secondary cell wall xylan synthesis complexes.
Secondarily, the protein interaction work on the cellulose synthase complex has also led to progress beyond the state of the art. The results of the affinity purification of the complex have pointed towards novel protein interactors involved in the cellulose synthesis complex that are currently investigated in detail by collaborators.
Tertiary, and most significantly, based on the project’s progress we believe that the methodology of size exclusion chromatography with SWATH acquisition applied on Arabidopsis leads to a significant understanding of plant biosynthetic machineries and eventually of their activities. This will deepen our current understanding of the plant molecular biology, especially on systems level, in order to develop crops that are resistant to environmental challenges, as well as varieties of plants for production of biofuels. This is a challenge that has enormous socio-economic impact.