Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (ß-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.

Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation.

Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.

Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.

The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 µg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.

Cytological analysis and structural quantification of FtsZ1-2 and FtsZ2-1 network characteristics in Physcomitrella patens.

Although the concept of the cytoskeleton as a cell-shape-determining scaffold is well established, it remains enigmatic how eukaryotic organelles adopt and maintain a specific morphology. The Filamentous Temperature Sensitive Z (FtsZ) protein family, an ancient tubulin, generates complex polymer networks, with striking similarity to the cytoskeleton, in the chloroplasts of the moss Physcomitrella patens. Certain members of this protein family are essential for structural integrity and shaping of chloroplasts, while others are not, illustrating the functional diversity within the FtsZ protein family. Here, we apply a combination of confocal laser scanning microscopy and a self-developed semi-automatic computational image analysis method for the quantitative characterisation and comparison of network morphologies and connectivity features for two selected, functionally dissimilar FtsZ isoforms, FtsZ1-2 and FtsZ2-1. We show that FtsZ1-2 and FtsZ2-1 networks are significantly different for 8 out of 25 structural descriptors. Therefore, our results demonstrate that different FtsZ isoforms are capable of generating polymer networks with distinctive morphological and connectivity features which might be linked to the functional differences between the two isoforms. To our knowledge, this is the first study to employ computational algorithms in the quantitative comparison of different classes of protein networks in living cells.

Approaches to Characterize Organelle, Compartment, or Structure Purity.

The function of subcellular structures is defined by their specific sets of proteins, making subcellular protein localization one of the most important topics in organelle research. To date, many organelle proteomics workflows involve the (partial) purification of the desired subcellular structure and the subsequent analysis of the proteome using tandem mass spectrometry (MS/MS). This chapter gives an overview of the methods that have been used to assay the purity and enrichment of subcellular structures, with an emphasis on quantitative proteomics using differently enriched subcellular fractions. We introduce large-scale-based criteria for assignment of proteins to subcellular structures and describe in detail the use of 15N metabolic labeling in moss to characterize plastid and mitochondrial proteomes.

The mitochondrial proteome of the moss Physcomitrella patens.

Extant basal land plants are routinely used to trace plant evolution and to track strategies for high abiotic stress resistance. Whereas the structure of mitochondrial genomes and RNA editing are already well studied, mitochondrial proteome research is restricted to a few data sets. While the mitochondrial proteome of the model moss Physcomitrella patens is covered to an estimated 15-25% by proteomic evidence to date, the available data have already provided insights into the evolution of metabolic compartmentation, dual targeting and mitochondrial heterogeneity. This review summarizes the current knowledge about the mitochondrial proteome of P. patens, and gives a perspective on its use as a mitochondrial model system. Its amenability to gene editing, metabolic labelling as well as fluorescence microscopy provides a unique platform to study open questions in mitochondrial biology, such as regulation of protein stability, responses to stress and connectivity to other organelles. Future challenges will include improving the proteomic resources for P. patens, and to link protein inventories and modifications as well as evolutionary differences to the functional level.

Mitochondrial Dynamics and the ER: The Plant Perspective.

Whereas contact sites between mitochondria and the ER have been in the focus of animal and fungal research for several years, the importance of this organellar interface and the molecular effectors are largely unknown for plants. This work gives an introduction into known evolutionary differences of molecular effectors of mitochondrial dynamics and interactions between animals, fungi, and plants. Using the model plant Physcomitrella patens, we provide microscopic evidence for the existence of mitochondria-ER interactions in plants and their correlation with mitochondrial constriction and fission. We further investigate a previously identified protein of unknown function (MELL1), and show that it modulates the amount of mitochondrial association to the ER, as well as mitochondrial shape and number.

Evolution and communication of subcellular compartments: An integrated approach.

Compartmentation is a fundamental feature of eukaryotic cells and the basis for metabolic complexity. We recently reported on the protein compartmentation in the moss Physcomitrella patens. This study utilized a combination of quantitative proteomics, comparative genomics, and single protein tagging and provided data on the postendosymbiotic evolution of plastids and mitochondria, on organellar communication, as well as on inter- and intracellular heterogeneity of organelles. We highlight potential organelle interaction hubs with specific protein content such as plastid stromules, and report on the plasticity of protein targeting to organelles.

Targeted gene knockouts reveal overlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division, chloroplast shaping, cell patterning, plant development, and gravity sensing.

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in P. patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss P. patens.