Theme leaders: Bert Poolman (RUG) and Peter Verhaert (DUT)

Background

In the Netherlands, the Kluyver Centre is studying the industrial applicability of micro-organisms. Within the Kluyver Center there is a growing need to analyze the proteome biology of micro-organisms. In NPCI collaborative studies have been performed. In the Netherlands, the University of Groningen, is a leading centre for research on biocatalysis, cell physiology and membrane biology in microorganisms, with additional unique expertise in membrane proteomics and structural genomics. Microbiology in Utrecht is particular strong in cell-surface proteins and secretory proteins. Advanced proteomics techniques form an essential part of the research ambitions of the Kluyver Centre. Building on the productive collaboration with NPCI, we have agreed on a joint program for optimal alignment of innovative developments within NPC with the application of advanced tools in the Kluyver Centre’s research programs. We will collaborate by jointly generating data to understand the biology of selected model systems; fungi (most notably S. cerevisiae) and lactic acid bacteria are the main targets for our research. In coordination with the Kluyver Centre, NPC will invest significantly in “enabling technologies” that are essential for the Kluyver Centre’s research portfolio. At DUT priority is given to development and implementation of peptidomics technology. A second priority concerns shared investment by Kluyver Centre and NPC in sophisticated equipment for the development of single-cell analysis systems (DUT) and organelle proteomics technology (RUG). For routine proteomics analyses, the Kluyver Centre partners will invest in equipment and personnel in-house, using the NPC Hotels for state-of-the-art support. Key areas to be addressed in the coming years concern the time and space resolved proteomic analysis of the cell, i.e. the proteome as a function of the cell cycle and resolving proteins in different parts of the cell (Poolman, Veenhoff, van der Klei, Wösten). Although protocols for the isolation and purification of yeast organelles (nuclei, vacuoles and peroxisomes) have been developed (Poolman, Veenhoff, van der Klei), the next challenge will be to reach a similar level of purity and proteomic detail for any point in the cell cycle. Also, it will be a challenge to apply technology developed for yeast to other fungi (Verhaert, Wösten). The implementation of Fluorescence Activated Cell Sorting (FACS) proteomics studies is a technical development in which subpopulations within cultures may be discriminated (van der Klei). Further technical advances at the proteomics, peptidomics and bioinformatics level are needed (Heck, Poolman, Veenhoff, Verhaert).

The main ambitions of this theme are to establish timeresolved (progression through cell cycle) and spaceresolved (organellar and cell type resolution) proteomics in Yeast and to integrate proteomics and peptidomics in the elucidation of protein turnover and the discovery of secreted peptides in fungi.

Approach

A peptidomics platform will be established (Verhaert, Pronk), aimed at screening for these low molecular weight compounds in culture media of various industrially relevant micro-organisms. The proposed platform will comprise (multidimensional) nanoflow liquid-chromatography technology and high mass accuracy modern (tandem) mass spectrometry. In order to specifically detect and quantify signalling molecules, (differential) peptide display technology will be combined with multiplexed isobaric mass tagging. While still technically challenging, peptidome analyses can be used to identify peptides released by cellular proteolysis and, therefore, to investigate protein degradation. One of the project goals is to investigate the potential of integrating proteomic and peptidomic analysis for the determination of protein turn-over (Verhaert, Wösten, Pronk, Heck). The filamentous fungus Penicillium will be developed further as efficient cell factory for the production of complex peptides, i.e., peptide antibiotics. Wösten and van der Klei will compare the proteome of P. chrysogenum hyphal tip cells relative to that of sub-apical cells, using laser microdissection methods.

Proteomic studies of filamentous fungi are scarce, despite the prevalence of these organisms in the industry, and their importance as both human and plant pathogens. On the other hand, there is wealth of information on the various subproteomes (organelles, complexes) of S. cerevisiae. The methodology developed within NPCI will now be applied in other fungi. Proteomics experiments become significantly more interesting and informative if an accurate description of the sample in terms of “time” and “space” is available. Within NPC2 there will be strong focus on the dynamic nature of yeast organelles, in particular the nucleus. Cell cycle-dependent proteomics of nuclei, nuclear envelopes and specific nuclear function related sub-complexes, such as those of the nuclear transport chaperones, will be carried out (Veenhoff, Poolman, Krijgsveld). The combination of proteomics with confocal fluorescence microscopy (time-lapse imaging, FRAP measurements), allows a high resolution description of nuclear events in terms of localization and composition.

Deliverables

• Methods for the isolation of nuclei and nuclear envelopes; microbodies throughout the cell cycle;
• Faster methods for the isolation of nuclei to allow proteomics of ‘labile’ / dynamic complexes;
• FACS-based proteomics to discriminate subpopulations within a culture;
• Calalogue of the microbody proteome of Penicillium chrysogenum;
• Catalogue of the secretome of Aspergillus niger under different growth condition;
• Pulse chase labelling procedures to monitor protein turn over in filamentous fungi;
• Quantified proteomic data of yeast nuclei & nuclear envelopes from defined stages of cell cycle
• Fluorescent tags for labeling of proteins in situ, i.e. methods alternative to GFP tagging;
• Proof-of-concept that peptides from industrially important microorganisms can be detected, identified;
• Integrated qualitative and quantitative MS-based peptide analytics into a world-class peptidomics facility suitable for highly sensitive peptide analysis of any biological source/origin;