Deep Proteomics reveals incorporation of unedited proteins into mitochondrial protein complexes in Arabidopsis.

The mitochondrial proteome consists of numerous types of proteins which either are encoded and synthesized in the mitochondria, or encoded in the cell nucleus, synthesized in the cytoplasm and imported into the mitochondria. Their synthesis in the mitochondria, but not in the nucleus, relies on the editing of the primary transcripts of their genes at defined sites. Here, we present an in-depth investigation of the mitochondrial proteome of Arabidopsis (Arabidopsis thaliana) and a public online platform for the exploration of the data. For the analysis of our shotgun proteomic data, an Arabidopsis sequence database was created comprising all available protein sequences from the TAIR10 and Araport11 databases, supplemented with sequences of proteins translated from edited and non-edited transcripts of mitochondria. Amino acid sequences derived from partially edited transcripts were also added to analyze proteins encoded by the mitochondrial genome. Proteins were digested in parallel with six different endoproteases to obtain maximum proteome coverage. The resulting peptide fractions were finally analyzed using liquid chromatography (LC) coupled to ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS). We generated a 'deep mitochondrial proteome' of 4,692 proteins. 1,339 proteins assigned to mitochondria by the SUBA5 database (https://suba.live) accounted for >80% of the total protein mass of our fractions. The coverage of proteins by identified peptides was particularly high compared to single-protease digests, allowing the exploration of differential splicing and RNA editing events at the protein level. We show that proteins translated from non-edited transcripts can be incorporated into native mitoribosomes and the ATP synthase complex. We present a portal for the use of our data, based on 'proteomaps' with directly linked protein data. The portal is available at www.proteomeexplorer.de.

The Viscum album Gene Space database.

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on 'Benchmarking Universal Single-Copy Orthologs' (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA).

The gene space of European mistletoe (Viscum album).

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5' and 3' non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.

Structure and function of complex I in animals and plants - a comparative view.

The mitochondrial NADH dehydrogenase complex (complex I) has a molecular mass of about 1000 kDa and includes 40-50 subunits in animals, fungi and plants. It is composed of a membrane arm and a peripheral arm and has a conserved L-like shape in all species investigated. However, in plants and possibly some protists it has a second peripheral domain which is attached to the membrane arm on its matrix exposed side at a central position. The extra domain includes proteins resembling prokaryotic gamma-type carbonic anhydrases. We here present a detailed comparison of complex I from mammals and flowering plants. Forty homologous subunits are present in complex I of both groups of species. In addition, five subunits are present in mammalian complex I, which are absent in plants, and eight to nine subunits are present in plant complex I which do not occur in mammals. Based on the atomic structure of mammalian complex I and biochemical insights into complex I architecture from plants we mapped the species-specific subunits. Interestingly, four of the five animal-specific and five of the eight to nine plant-specific subunits are localized at the inner surface of the membrane arm of complex I in close proximity. We propose that the inner surface of the membrane arm represents a workbench for attaching proteins to complex I, which are not directly related to respiratory electron transport, like nucleoside kinases, acyl-carrier proteins or carbonic anhydrases. We speculate that further enzyme activities might be bound to this micro-location in other groups of organisms.

The mitochondrial complexome of Arabidopsis thaliana.

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein-protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot-gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re-annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource (https://complexomemap.de/at_mito_leaves) to deposit the data and to allow straightforward access and custom data analyses.

The 'protein complex proteome' of chloroplasts in Arabidopsis thaliana.

Here, a first GelMap of the chloroplast "protein complex proteome" of Arabidopsis thaliana is presented. The GelMap software tool allows assigning multiple proteins to gel spots, thereby taking advantage of the high sensitivity of state-of-the-art mass spectrometry systems. Furthermore, the software allows functional annotation of all identified proteins. If applied to a 2D blue native (BN)/SDS gel, GelMap can selectively display protein complexes of low abundance. For the chloroplast GelMap, highly purified organelles were separated by 2D BN/SDS PAGE and spots were automatically detected using Delta 2D software. Within 287 spots, a total of 1841 proteins were identified (on average 6.4 proteins per spot), representing a set of 436 non redundant proteins. Most of these proteins form part of protein complexes. The quality of the map is reflected by its inclusion of a more or less complete set of protein complexes described for chloroplasts in the literature. The GelMap is publically available at www.gelmap.de/arabidopsis-chloro and may be used as a resource for identifying novel protein complexes within any of its functional categories. BIOLOGICAL SIGNIFICANCE: The chloroplast GelMap represents a data resource for the definition of protein complexes in the model plant A. thaliana. It should be useful for in depth understanding of chloroplast biochemistry, as illustrated by the discovery of so far unknown protein complexes. The GelMap is publically available at www.gelmap.de/arabidopsis-chloro.

Functional Annotation of 2D Protein Maps: The GelMap Portal.

In classical proteome analyses, final experimental data are (a) images of 2D protein separations obtained by gel electrophoresis and (b) corresponding lists of proteins which were identified by mass spectrometry (MS). For data annotation, software tools were developed which allow the linking of protein identity data directly to 2D gels ("clickable gels"). GelMap is a new online software tool to annotate 2D protein maps. It allows (i) functional annotation of all identified proteins according to biological categories defined by the user, e.g., subcellular localization, metabolic pathway, or assignment to a protein complex and (ii) annotation of several proteins per analyzed protein "spot" according to MS primary data. Options to differentially display proteins of functional categories offer new opportunities for data evaluation. For instance, if used for the annotation of 2D Blue native/SDS gels, GelMap allows the identification of protein complexes of low abundance. A web portal has been established for presentation and evaluation of protein identity data related to 2D gels and is freely accessible at http://www.gelmap.de/.

Defining the protein complex proteome of plant mitochondria.

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.

GelMap-a novel software tool for building and presenting proteome reference maps.

Protein separation by two-dimensional gel electrophoresis is of central importance for proteomics. Upon combination with systematic protein identifications by mass spectrometry, large data sets are routinely generated in several proteome laboratories which can be used as "reference maps" for future analyses of analogous biochemical fractions. Here we present GelMap, a novel software tool for the building presentation and evaluation of proteomic reference maps. Variable frames are introduced in order to group proteins into functional categories on three levels or into categories according to differential abundance during comparative proteome analyses. The software is easy to handle as it only requires uploading two digital files to a web site. An additional file including detailed information on all proteins can be combined with the primary map. Two different gel-based projects are presented to illustrate the capacity of GelMap for proteome annotation and evaluation.