System-wide analysis of absolute protein abundances during the development from spores to multinucleated hyphae of the filamentous fungus "Ashbya gossypii"

The biological system: Ashbya gossypii is a unique organism to study dramatic evolutionary changes within rather short time scales. Phylogenetically, it is closely related to the yeast Saccharomyces cerevisiae, which proliferates by budding of uninucleated oval cells. In contrast, A. gossypii carrying an almost identical set of genes proliferates by continuous elongation of multinucleated long cells, called hyphae, which frequently branch to form new hyphae but never divide. This generates a fast spreading fungal mycelium, which can cover the surface of an agar plate within a week starting from a single spore. Nutrient limitations in the center of the mycelium induce formation of spores, which can be isolated with high purity. The goal of this PhD thesis was to characterize the proteome at different stages of this rather simple life cycle at a resolution and quantification level so far not achieved with other filamentous fungi.
Chapter1 describes the principle of the methods used to analyze peptide mixtures obtained by exhaustive digestions of cell extracts with trypsin. Tandem mass spectrometry (LC–MS/MS) was performed with a reference peptide library extracted from the A. gossypii genome. Up to 3,900 proteins of the predicted 4,748 proteins were detected using stringent statistical analyses, and their relative abundances were estimated using two methods. In several cases heavy isotope-labeled synthetic AQUA peptides of known concentration were spiked prior to the MS runs to allow absolute quantifications. Experiments to determine the changing protein compositions and phophorylations during spore germination were performed with biological and technical replicas. For experiments aiming at highest possible resolution the peptide mixtures were prefractionated and each fraction separately analyzed. In total 102 MS runs were performed. The results of most experiments are summarized as tables in the Appendix. Only the high resolution experiments of peptide mixtures (spiked with 40 AQUA peptides) of spores, germ bubbles, and young mycelia are documented and discussed in detail in this PhD thesis.
Chapter2 documents in three sub-chapters first the preparations of tryptic peptides from spores and the identification of 3,895 proteins, second the relative abundances of these proteins, and third the approaches used to assign copy numbers per spore to each identified protein. In total, a single A. gossypii spore contains 40x106 proteins from one copy to several million copies. Special attention was given to the histone proteome including posttranslational modifications. The nucleus in the middle of each spore carries a haploid genome of 9x106bp. A genome of this size is associated with approx. 50,000 nucleosomes predicting close to 100,000 copies of each histone per spore. Using AQUA peptides as reference 34,000 copies of histones H3 and H4 were determined confirming that the calculated copy numbers of the other proteins are fairly close to the real copy numbers.
Chapter3 discusses the approach to determine the proteome of isotropically expending germ bubbles. Germination proceeds asynchronously and only mixtures of germ bubbles with different sizes plus morphological unaltered spores can be isolated. Only 5 hours after spore inoculation in liquid medium this mixture consists of 35% germ bubbles and 65% spores. The mixed protein composition was determent and 65% of the known copy numbers in spores were subtracted. The obtained copy numbers for the close to 3,700 proteins most likely reflect the proteome of an average germ bubble, which has not started polar growth yet. In total, 80x106 proteins are present in each germ bubble. The adjustment of copy numbers by subtracted of 65% of spore specific proteins resulted in negative numbers in 200 cases strongly indicating that this proteins are actively degraded during the pre-germination phase.
Chapter4 focuses on the proteome of an average young mycelium formed after 11 hours of spore inoculation. Each young mycelium is a giant cell with multiple polar growth sites (hyphal tips) and multiple nuclei. The population of these giant cells is heterogeneous, the number of growing hyphal tips ranges from 3 to 20, the total length of the network of hyphae ranges from 19 to 400 µm, and the number of nuclei ranges from 10 to over 200. We defined the mean of 200 measured mycelia as standard young mycelium, which has seven polarity axes with a total length of 120 µm and contains 48 nuclei. The analyzed cell mixture of young mycelia still contains 15% of spores and germ bubbles the protein compositions of which were neglected because their biomass reflect less the 1% of the total biomass of the growing young mycelia. A standard young mycelium contains 2x109 proteins the largest class consisting of ribosomal proteins.
Chapter5 represents the dynamic of the A. gossypii proteome during development from spores to young mycelia. Importantly, the contribution of proteins to the total biomass increases from 5% to 15% during this development and remains constant thereafter. Two different types of clustering analyses revealed 800 proteins substantially increased in abundance. Only 200 proteins were degraded during the development and the rest changed marginally or showed up- shifts followed by down-shifts or vice versa. For the septin complex we could show that the relative protein abundances are in the same stoichiometric range and the protein copy numbers have a uniform behavior during development.