Organellar Proteomics Research Articles

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Identification of Neighboring Proteins of Endosomal Regulators by Using TurboID-Based Proximity Labeling.

In the endomembrane system, multivesicular bodies (MVBs) play a crucial role in sorting ubiquitinated membrane proteins into intraluminal vesicles for degradation upon fusion with vacuoles or lysosomes. This process involves regulations by multiprotein complexes, including endosomal sorting complex required for transport (ESCRT) I-III, and accessory proteins. Although many organellar proteomes have been identified in plant cells, the information of specific proteomes associated with regulators engaged in MVB biogenesis remains limited. Here, using the ESCRT component FREE1 as an example, we describe a method to identify neighboring proteins of endosomal regulators by using an approach of TurboID-based proximity labeling.

Chloroplast protein translocation pathways and ubiquitin-dependent regulation at a glance.

Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.

Isolation and Quality Control of Yeast Mitochondria.

The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containingiron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.

Functional coupling of presequence processing and degradation in human mitochondria.

The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.

The Phytotoxicity of Meta-Tyrosine Is Associated With Altered Phenylalanine Metabolism and Misincorporation of This Non-Proteinogenic Phe-Analog to the Plant's Proteome.

Plants produce a myriad of specialized (secondary) metabolites that are highly diverse chemically, and exhibit distinct biological functions. Here, we focus on meta-tyrosine (m-tyrosine), a non-proteinogenic byproduct that is often formed by a direct oxidation of phenylalanine (Phe). Some plant species (e.g., Euphorbia myrsinites and Festuca rubra) produce and accumulate high levels of m-tyrosine in their root-tips via enzymatic pathways. Upon its release to soil, the Phe-analog, m-tyrosine, affects early post-germination development (i.e., altered root development, cotyledon or leaf chlorosis, and retarded growth) of nearby plant life. However, the molecular basis of m-tyrosine-mediated (phyto)toxicity remains, to date, insufficiently understood and are still awaiting their functional characterization. It is anticipated that upon its uptake, m-tyrosine impairs key metabolic processes, or affects essential cellular activities in the plant. Here, we provide evidences that the phytotoxic effects of m-tyrosine involve two distinct molecular pathways. These include reduced steady state levels of several amino acids, and in particularly altered biosynthesis of the phenylalanine (Phe), an essential α-amino acid, which is also required for the folding and activities of proteins. In addition, proteomic studies indicate that m-tyrosine is misincorporated in place of Phe, mainly into the plant organellar proteomes. These data are supported by analyses of adt mutants, which are affected in Phe-metabolism, as well as of var2 mutants, which lack FtsH2, a major component of the chloroplast FtsH proteolytic machinery, which show higher sensitivity to m-tyrosine. Plants treated with m-tyrosine show organellar biogenesis defects, reduced respiration and photosynthetic activities and growth and developmental defect phenotypes.

Development of a Photoactivatable Proximity Labeling Method for the Identification of Nuclear Proteins

The precise characterization of organellar proteomes is essential for deciphering a variety of cellular functions at the molecular level. We here report a photoactivatable proximity labeling method...

Extracellular Matrix Proteome: Isolation of ECM Proteins for Proteomics Studies.

Understanding molecular mechanisms and cellular metabolism in varied plant processes necessitates knowledge of the expressed proteins and their subcellular distribution. Spatial partitioning of organelles generates an enclosed milieu for physiochemical reactions designed and tightly linked to a specific organelle function. Of which, extracellular matrix (ECM)/cell wall (CW) is a dynamic and chemically active compartment. The ECM proteins are organized into complex structural and functional networks involved in several metabolic processes, including carbon and nitrogen metabolism. Organellar proteomics aim for comprehensive identification of resident proteins that rely on the isolation of highly purified organelle free from contamination by other intracellular components. Extraction and isolation of plant ECM proteins features key caveats due to the lack of adjoining membrane, the presence of a polysaccharide-protein network that traps contaminants, and the existence of high phenolic content. Furthermore, due to diverse biochemical forces, including labile, weakly bound and strongly bound protein in the protein-polysaccharide matrix different elution procedures are required to enrich ECM proteins. Here, we describe a method that allows efficient fractionation of plant ECM, extraction of ECM proteins and protein profiling from variety of crop plants, including rice, chickpea and potato. This method can easily be adapted to other plant species for varied experimental conditions.

Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID.

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells

ABSTRACTThe double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

AAA Proteases: Guardians of Mitochondrial Function and Homeostasis.

Mitochondria are dynamic, semi-autonomous organelles that execute numerous life-sustaining tasks in eukaryotic cells. Functioning of mitochondria depends on the adequate action of versatile proteinaceous machineries. Fine-tuning of mitochondrial activity in response to cellular needs involves continuous remodeling of organellar proteome. This process not only includes modulation of various biogenetic pathways, but also the removal of superfluous proteins by adenosine triphosphate (ATP)-driven proteolytic machineries. Accordingly, all mitochondrial sub-compartments are under persistent surveillance of ATP-dependent proteases. Particularly important are highly conserved two inner mitochondrial membrane-bound metalloproteases known as m-AAA and i-AAA (ATPases associated with diverse cellular activities), whose mis-functioning may lead to impaired organellar function and consequently to development of severe diseases. Herein, we discuss the current knowledge of yeast, mammalian, and plant AAA proteases and their implications in mitochondrial function and homeostasis maintenance.

Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins

Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.

Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle

The highly progressive neuromuscular disorder dystrophinopathy is triggered by primary abnormalities in the Dmd gene, which causes cytoskeletal instability and loss of sarcolemmal integrity. Comparative organellar proteomics was employed to identify sarcolemma-associated proteins with an altered concentration in dystrophic muscle tissue from the mdx-4cv mouse model of dystrophinopathy. A lectin agglutination method was used to prepare a sarcolemma-enriched fraction and resulted in the identification of 190 significantly changed protein species. Proteomics established differential expression patterns for key components of the muscle plasma membrane, cytoskeletal network, extracellular matrix, metabolic pathways, cellular stress response, protein synthesis, immune response and neuromuscular junction. The deficiency in dystrophin and drastic reduction in dystrophin-associated proteins appears to trigger (i) enhanced membrane repair involving myoferlin, dysferlin and annexins, (ii) increased protein synthesis and the compensatory up-regulation of cytoskeletal proteins, (iii) the decrease in the scaffolding protein periaxin and myelin PO involved in myelination of motor neurons, (iv) complex changes in bioenergetic pathways, (v) elevated levels of molecular chaperones to prevent proteotoxic effects, (vi) increased collagen deposition causing reactive myofibrosis, (vii) disturbed ion homeostasis at the sarcolemma and associated membrane systems, and (viii) a robust inflammatory response by the innate immune system in response to chronic muscle damage. SignificanceDuchenne muscular dystrophy is a devastating muscle wasting disease and represents the most frequently inherited neuromuscular disorder in humans. Genetic abnormalities in the Dmd gene cause a loss of sarcolemmal integrity and highly progressive muscle fibre degeneration. Changes in the neuromuscular system are associated with necrosis, fibrosis and inflammation. In order to evaluate secondary changes in the sarcolemma membrane system due to the lack of the membrane cytoskeletal protein dystrophin, comparative organellar proteomics was used to study the mdx-4cv mouse model of dystrophinopathy. Mass spectrometric analyses identified a variety of altered components of the extracellular matrix-sarcolemma-cytoskeleton axis in dystrophic muscles. This included proteins involved in membrane repair, cytoskeletal restoration, calcium homeostasis, cellular signalling, stress response, neuromuscular transmission and reactive myofibrosis, as well as immune cell infiltration. These pathobiochemical alterations agree with the idea of highly complex secondary changes in X-linked muscular dystrophy and support the concept that micro-rupturing of the dystrophin-deficient plasma membrane is at the core of muscle wasting pathology.

Fungal Peroxisomes Proteomics.

Peroxisomes in fungi are involved in a huge number of different metabolic processes. In addition, non-metabolic functions have also been identified. The proteins that are present in a particular peroxisome determine its metabolic function, whether they are the matrix localized enzymes of the different metabolic pathways or the membrane proteins involved in transport of metabolites across the peroxisomal membrane. Other peroxisomal proteins play a role in organelle biogenesis and dynamics, such as fission, transport and inheritance. Hence, obtaining a complete overview of which proteins are present in peroxisomes at a given time or under a given growth condition provides invaluable insights into peroxisome biology. Bottom up approaches are ideal to follow one or a few proteins at a time but they are not able to give a global view of the content of peroxisomes. To gain such information, top down approaches are required and one that has provided valuable insights into peroxisome function is mass spectrometry based organellar proteomics. Here, we discuss the findings of several such studies in yeast and filamentous fungi and outline new insights into peroxisomal function that were gained from these studies.

Nuclear Proteome: Isolation of Intact Nuclei, Extraction of Nuclear Proteins, and 2-DE Analysis.

Proteome profiling aims to unravel the mystery of biological complexity encoded by the genome. The successful proteome profiling largely depends upon analytical approaches because single-step proteome characterization of eukaryotic cells is difficult due to the large number of proteins expressed and their complex physiochemical properties. Organellar proteomics helps in identifying a refined set of proteins by pinpointing certain activities to specific organelles, thereby increasing our knowledge of cellular processes. The reliability of a plant organelle proteome is intimately dependent on the purity of the organelle preparation. Methodological improvements in sample handling, organelle fractionation, and protein extraction are therefore crucial to plant subcellular proteomics. The nuclear proteins are organized into complex regulatory networks and perform varied cellular functions. Therefore, characterization of the nuclear proteome is an important step toward accumulating knowledge about regulation of gene expression and function. In this chapter, we present methods for the isolation of nuclei, purification of nuclear proteins, and proteome profiling that have been adapted for proteomic characterization of economically important crop species, such as chickpea.

Massive Protein Import into the Early-Evolutionary-Stage Photosynthetic Organelle of the Amoeba Paulinella chromatophora

The endosymbiotic acquisition of mitochondria and plastids more than 1 Ga ago profoundly impacted eukaryote evolution. At the heart of understanding organelle evolution is the re-arrangement of the endosymbiont proteome into a host-controlled organellar proteome. However, early stages in this process as well as the timing of events that underlie organelle integration remain poorly understood. The amoeba Paulinella chromatophora contains cyanobacterium-derived photosynthetic organelles, termed "chromatophores," that were acquired more recently (around 100 Ma ago). To explore the re-arrangement of an organellar proteome during its integration into a eukaryotic host cell, here we characterized the chromatophore proteome by protein mass spectrometry. Apparently, genetic control over the chromatophore has shifted substantially to the nucleus. Two classes of nuclear-encoded proteins-which differ in protein length-are imported into the chromatophore, most likely through independent pathways. Long imported proteins carry a putative, conserved N-terminal targeting signal, and many specifically fill gaps in chromatophore-encoded metabolic pathways or processes. Surprisingly, upon heterologous expression in a plant cell,the putative chromatophore targeting signal conferred chloroplast localization. This finding suggests common features in the protein import pathways of chromatophores and plastids, two organelles that evolved independently and more than 1Ga apart from each other. By combining experimental data with insilico predictions, we provide a comprehensive catalog of almost 450 nuclear-encoded, chromatophore-targeted proteins. Interestingly, most imported proteins seem to derive from ancestral host genes, suggesting that the re-targeting of nuclear-encoded proteins that resulted from endosymbiotic gene transfers plays only a minor role at the onset of chromatophore integration.

Functional characterization of the lysosomal membrane protein TMEM192 in mice.

The Transmembrane protein 192 (TMEM192) is a lysosomal/late endosomal protein initially discovered by organellar proteomics. TMEM192 exhibits four transmembrane segments with cytosolic N- and C-termini and forms homodimers. Devoid of significant homologies, the molecular function of TMEM192 is currently unknown. Upon TMEM192 knockdown in hepatoma cells, a dysregulation of autophagy and increased apoptosis were reported. Here, we aimed to define the physiological role of TMEM192 by analysing consequences of TMEM192 ablation in mice. Therefore, we compared the biochemical properties of murine TMEM192 to those of the human orthologue. We reveal lysosomal residence of murine TMEM192 and demonstrate ubiquitous tissue expression. In brain, TMEM192 expression was pronounced in the hippocampus but also present in the cortex and cerebellum, as analysed based on a lacZ reporter allele. Murine TMEM192 undergoes proteolytic processing in a tissue-specific manner. Thereby, a 17 kDa fragment is generated which was detected in most murine tissues except liver. TMEM192 processing occurs after lysosomal targeting by pH-dependent lysosomal proteases. TMEM192-/- murine embryonic fibroblasts (MEFs) exhibited a regular morphology of endo-/lysosomes and were capable of performing autophagy and lysosomal exocytosis. Histopathological, ultrastructural and biochemical analyses of all major tissues of TMEM192-/- mice demonstrated normal lysosomal functions without apparent lysosomal storage. Furthermore, the abundance of the major immune cells was comparable in TMEM192-/- and wild type mice. Based on this, we conclude that under basal conditions in vivo the loss of TMEM192 can be efficiently compensated by alternative pathways. Further studies will be required to decipher its molecular function.