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Plants are essential for life and are extremely diverse organisms with unique molecular capabilities 1 . Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana . Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions. A quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana provides a valuable resource for plant research.

Acute protein folding stress in the mitochondrial matrix activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition. Mammalian mitochondrial stress responses The mitochondrial unfolded protein response (UPR mt ) pathway has been studied in detail in the Caenorhabditis elegans roundworm, where it has been shown to sense protein misfolding within the mitochondrial matrix and to induce a program of nuclear gene expression to counteract this stress. How mammalian cells respond to unfolded matrix proteins has remained much less clear. Christian Münch and Wade Harper used pharmacological inhibitors to induce acute protein folding stress in the mitochondrial matrix, and performed transcriptional and quantitative proteomic analysis to examine the response of mammalian cells. They observed widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. This was accompanied by a rapid reduction in the matrix-localized protein synthesis through translational inhibition. The work could spur further investigation of the mammalian UPR mt . The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans , the mitochondrial unfolded protein response (UPR mt ) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis 1 , 2 , 3 . While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression 4 , 5 , 6 , our understanding of mammalian UPR mt is rudimentary 7 , reflecting a lack of acute triggers for UPR mt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPR mt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8 ) or LON protease 9 , which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPR mt . This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10 ). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPR mt .

Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored. Here we monitor mitochondrial and nuclear gene expression in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. We show that nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, mitochondrial and cytosolic translation are rapidly, dynamically and synchronously regulated. Furthermore, cytosolic translation processes control mitochondrial translation unidirectionally. Thus, the nuclear genome coordinates mitochondrial and cytosolic translation to orchestrate the timely synthesis of OXPHOS complexes, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for studies of global gene regulation in mitochondria. The genes encoding the subunits of oxidative phosphorylation complexes are split between the nuclear and mitochondrial genomes, but their translation is synchronized by signalling from the cytosol to the mitochondria. Aligning mitochondrial and nuclear expression The OXPHOS (oxidative phosphorylation) complexes within the mitochondrial inner membrane generate the large majority of the cell's energy through the synthesis of ATP from ADP and inorganic phosphate during the oxidation of NADH by molecular oxygen. As the OXPOS complex contains subunits encoded by both the nuclear and the mitochondrial genomes, it has been widely assumed that there must be communication between the two compartments to coordinate gene expression. Stirling Churchman and colleagues have now characterized synthesis of the OXPHOS subunits. They find that nuclear and mitochondrial transcription programs are independently regulated under the direction of the nuclear genome. Regulation occurs not at the level of transcription, but rather in terms of translation, with mitochondrial translation regulated through the cytosolic ribosomes.

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein–protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice. The effect of natural genetic diversity on the proteome is characterized using an outbred mouse model with extensive variation; both transcripts and proteins from mouse livers are quantified to identify a large set of protein quantitative trait loci (pQTL), and mediation analysis identifies causal protein intermediates of distant pQTL. Natural genetic diversity and the proteome The investigation of how genetic variation governs gene expression has so far focused mainly on quantifying the impact on RNA transcripts. These authors have focused on protein and transcript abundance in a genetically diverse population of mice. In a proteome-wide analysis, they quantify transcripts and proteins from mouse livers and identify a large set of protein-level quantitative trait loci (pQTL). Mediation analysis identifies causal protein intermediates underlying distant pQTL, and hundreds of protein–protein associations.

Bacterial growth is crucially dependent on protein synthesis and thus on the cellular abundance of ribosomes and related proteins. Here, we show that the slow diffusion of the bulky tRNA complexes in the crowded cytoplasm imposes a physical limit on the speed of translation, which ultimately limits the rate of cell growth. To study the required allocation of ancillary translational proteins to alleviate the effect of molecular crowding, we develop a model for cell growth based on a coarse-grained partitioning of the proteome. We find that coregulation of ribosome- and tRNA-affiliated proteins is consistent with measured growth-rate dependencies and results in near-optimal allocation over a broad range of growth rates. The analysis further resolves a long-standing controversy in bacterial growth physiology concerning the growth-rate dependence of translation speed and serves as a caution against premature identification of phenomenological parameters with mechanistic processes.

Thaumarchaeota are among the most abundant microbial cells in the ocean, but to date, complete genome sequences for marine Thaumarchaeota are lacking. Here, we report the 1.23-Mbp genome of the pelagic ammonia-oxidizing thaumarchaeon “ Candidatus Nitrosopelagicus brevis” str. CN25. We present the first proteomic data, to our knowledge, from this phylum, which show a high proportion of proteins translated in oligotrophic conditions. Metagenomic fragment recruitment using data from the open ocean indicate the ubiquitous presence of Ca. N. brevis-like sequences in the surface ocean and suggest Ca . N. brevis as a model system for understanding the ecology and evolution of pelagic marine Thaumarchaeota. Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name “ Candidatus Nitrosopelagicus brevis” str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems 1 , 2 . Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress 3 – 5 . The abundance of ribosomal (r)-proteins (around 6% of the proteome; 10 7 copies per cell) 6 , 7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy 4 , 7 . However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited 7 . Here, we integrate quantitative global translatome and degradome proteomics 8 with genetically encoded Ribo–Keima 5 and Ribo–Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress. During nutrient stress, ribosomal protein abundance is regulated primarily by translational and non-autophagic degradative mechanisms, but ribosome density per cell is largely maintained by reductions in cell volume and rates of cell division.

Maternal infection and inflammation during pregnancy are associated with neurodevelopmental disorders in offspring, but little is understood about the molecular mechanisms underlying this epidemiologic phenomenon. Here, we leveraged single-cell RNA sequencing to profile transcriptional changes in the mouse fetal brain in response to maternal immune activation (MIA) and identified perturbations in cellular pathways associated with mRNA translation, ribosome biogenesis and stress signaling. We found that MIA activates the integrated stress response (ISR) in male, but not female, MIA offspring in an interleukin-17a-dependent manner, which reduced global mRNA translation and altered nascent proteome synthesis. Moreover, blockade of ISR activation prevented the behavioral abnormalities as well as increased cortical neural activity in MIA male offspring. Our data suggest that sex-specific activation of the ISR leads to maternal inflammation-associated neurodevelopmental disorders. This paper shows that maternal immune activation in mice induces changes in the mRNA translation machinery in the fetal brain and activates the integrated stress response in male fetuses, which mediates neurobehavioral abnormalities.

A large-scale analysis of variation in human protein levels between individuals is performed using mass-spectrometry-based proteomic technology, and a number of protein quantitative trait loci are identified; over 5% of proteins vary by more than 1.5-fold in their expression levels between individuals, and this variation is not always linked to RNA level. Control of human proteome variation Efforts to understand the mechanisms underlying phenotypic variation between individuals have focused mainly on events at the level of RNA and transcription factor binding, and on mapping the genetic loci responsible. Proteins are much closer to phenotypes than RNA but few studies have analysed protein variation on a global level. Here, a large-scale analysis of variation in protein levels between 95 diverse individuals genotyped in the HapMap Project is performed using mass spectrometry-based proteomic technology, and a number of protein quantitative trait loci are identified. Over 5% of proteins vary more than 1.5 fold in their expression levels between individuals, and this variation is not always linked to RNA levels. Gene expression differs among individuals and populations and is thought to be a major determinant of phenotypic variation. Although variation and genetic loci responsible for RNA expression levels have been analysed extensively in human populations 1 , 2 , 3 , 4 , 5 , our knowledge is limited regarding the differences in human protein abundance and the genetic basis for this difference. Variation in messenger RNA expression is not a perfect surrogate for protein expression because the latter is influenced by an array of post-transcriptional regulatory mechanisms, and, empirically, the correlation between protein and mRNA levels is generally modest 6 , 7 . Here we used isobaric tag-based quantitative mass spectrometry to determine relative protein levels of 5,953 genes in lymphoblastoid cell lines from 95 diverse individuals genotyped in the HapMap Project 8 , 9 . We found that protein levels are heritable molecular phenotypes that exhibit considerable variation between individuals, populations and sexes. Levels of specific sets of proteins involved in the same biological process covary among individuals, indicating that these processes are tightly regulated at the protein level. We identified cis -pQTLs (protein quantitative trait loci), including variants not detected by previous transcriptome studies. This study demonstrates the feasibility of high-throughput human proteome quantification that, when integrated with DNA variation and transcriptome information, adds a new dimension to the characterization of gene expression regulation.

Here, the selection of substrates by the protein–RNA complex known as the signal recognition particle (SRP) is investigated in the bacterium Escherichia coli , revealing that the SRP has a strong preference for hydrophobic transmembrane domains of inner membrane proteins. Specificity of the signal recognition particle As nascent proteins are generated by translating ribosomes, they are simultaneously targeted for translocation into the endoplasmic reticulum by a protein–RNA complex known as the signal recognition particle (SRP). Günter Kramer and colleagues investigate the nature of SRP substrates and how are they selected in a study of the SRP interactome in the bacterium Escherichia coli . They find that SRP almost exclusively targets hydrophobic transmembrane domains (TMDs) of inner membrane proteins, rejecting proteins of the outer membrane and the periplasmic space. Their data also contradict previous thinking that SRP associates with the first TMD to emerge from a ribosome, and show that in many proteins that will eventually span the membrane several times, the SRP binds several TMDs. In a related paper also in this issue of Nature , Judith Frydman and co-workers, studying yeast cells, find that the SRP preferentially binds substrates destined for secretion before they are fully translated — specifically, through the non-coding elements in their mRNA and before 'targeting signals' of these substrates are translated. Signal recognition particle (SRP) is a universally conserved protein–RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons 1 . The existence of parallel co- and post-translational transport pathways 2 , however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome–nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA–SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.