Organelle Proteomics Research Articles

Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei.

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.

Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function (“dark” genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

A subcellular proteome atlas of the yeast Komagataella phaffii.

ABSTRACTThe compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

Hansenula PolymorphaVac8: A Vacuolar Membrane Protein Required for Vacuole Inheritance and Nucleus-Vacuole Junction Formation

Saccharomyces cerevisiae Vac8 is a vacuolar membrane protein, which functions in vacuole inheritance and fusion, nucleus-vacuole junctions, autophagy and the cytoplasm-to-vacuole-targeting pathway. Here, we analyzed Vac8 of the yeast Hansenula polymorpha. We show that HpVac8 localizes to the vacuolar membrane and concentrates in patches at nucleus-vacuole junctions. Analysis of a VAC8 deletion strain indicated that HpVac8 is required for vacuole inheritance and the formation of nuclear-vacuole junctions, but not for vacuole fusion. Previously, organelle proteomics resulted in the identification of Vac8 in peroxisomal fractions isolated from H. polymorpha and S. cerevisiae. However, deletion of H. polymorpha VAC8 had no effect on peroxisome biogenesis or peroxisome-vacuole contact sites.

Membrane Transporters in Citrus clementina Fruit Juice-Derived Nanovesicles.

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D2O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way.

Origins, function, and regulation of the TOC-TIC general protein import machinery of plastids.

The evolution of chloroplasts from the original endosymbiont involved the transfer of thousands of genes from the ancestral bacterial genome to the host nucleus, thereby combining the two genetic systems to facilitate coordination of gene expression and achieve integration of host and organelle functions. A key element of successful endosymbiosis was the evolution of a unique protein import system to selectively and efficiently target nuclear-encoded proteins to their site of function within the chloroplast after synthesis in the cytoplasm. The chloroplast TOC-TIC (translocon at the outer chloroplast envelope-translocon at the inner chloroplast envelope) general protein import system is conserved across the plant kingdom, and is a system of hybrid origin, with core membrane transport components adapted from bacterial protein targeting systems, and additional components adapted from host genes to confer the specificity and directionality of import. In vascular plants, the TOC-TIC system has diversified to mediate the import of specific, functionally related classes of plastid proteins. This functional diversification occurred as the plastid family expanded to fulfill cell- and tissue-specific functions in terrestrial plants. In addition, there is growing evidence that direct regulation of TOC-TIC activities plays an essential role in the dynamic remodeling of the organelle proteome that is required to coordinate plastid biogenesis with developmental and physiological events.

MORF9 Functions in Plastid RNA Editing with Tissue Specificity.

RNA editing in plant mitochondria and plastids converts specific nucleotides from cytidine (C) to uridine (U). These editing events differ among plant species and are relevant to developmental stages or are impacted by environmental conditions. Proteins of the MORF family are essential components of plant editosomes. One of the members, MORF9, is considered the core protein of the editing complex and is involved in the editing of most sites in chloroplasts. In this study, the phenotypes of a T-DNA insertion line with loss of MORF9 and of the genetic complementation line of Arabidopsis were analyzed, and the editing efficiencies of plastid RNAs in roots, rosette leaves, and flowers from the morf9 mutant and the wild-type (WT) control were compared by bulk-cDNA sequencing. The results showed that most of the known MORF9-associated plastid RNA editing events in rosette leaves and flowers were similarly reduced by morf9 mutation, with the exception that the editing rate of the sites ndhB-872 and psbF-65 declined in the leaves and that of ndhB-586 decreased only in the flowers. In the roots, however, the loss of MORF9 had a much lower effect on overall plastid RNA editing, with nine sites showing no significant editing efficiency change, including accD-794, ndhD-383, psbZ-50, ndhF-290, ndhD-878, matK-706, clpP1-559, rpoA-200, and ndhD-674, which were reduced in the other tissues. Furthermore, we found that during plant aging, MORF9 mRNA level, but not the protein level, was downregulated in senescent leaves. On the basis of these observations, we suggest that MORF9-mediated RNA editing is tissue-dependent and the resultant organelle proteomes are pertinent to the specific tissue functions.

Mitochondrial Nascent Chain Quality Control Determines Organelle Form and Function

Proteotoxicity has long been considered a key factor in mitochondrial dysfunction and human disease. The origin of the endogenous offending toxic substrates and the regulatory pathways to deal with these insults, however, have remained unclear. Mitochondria maintain a compartmentalized gene expression system that in animals is only responsible for synthesis of 1% of the organelle proteome. Because of the relatively small contribution of the mitochondrial genome to the overall proteome, the synthesis and quality control of these nascent chains to maintain organelle proteostasis has long been overlooked. However, recent research has uncovered mechanisms by which defects to the quality control of mitochondrial gene expression are linked to a novel cellular stress response that impinges upon organelle form and function and cell fitness. In this review, we discuss the mechanisms for a key event in the response: activation of the metalloprotease OMA1. This severs the membrane tether of the dynamin-related GTPase OPA1, which is a critical determinant for mitochondrial morphology and function. We also highlight the evolutionary conservation from bacteria of these quality-control mechanisms to maintain membrane integrity, gene expression, and cell fitness.

Comparison of different methods for the isolation of Arabidopsis thaliana nuclear membranes

In animal and yeast system, subcellular fractionation has been widely used in studies of protein localization and organelle proteomics. Alternatively, it has not been an effective way of study in plant system because of some experimental limitations. The main aim of this study is to optimize subcellular and subnuclear fractionation of wild-type of Arabidopsis thaliana ecotype Colombia (Col-0) leaves by comparing three different methods to isolate nuclear membrane. In the study, we at first optimized nuclear washing steps to remove chloroplast contents from nuclear fractions. By optimizing speed of centrifugation and chemical component of the nuclear washing buffer, purified nuclear fractions was obtained. After measurement of protein amount for each fraction, purity of the fractions was analyzed by western blot assay with some specific cell compartment markers such as anti-Histone3 for nuclear fraction and anti-Rubisco for cytoplasmic fraction. Also, lactate dehydrogenase enzyme assay was used to confirm purity of the fractions. Then, subnuclear fractionation was done to isolate purified nuclear membrane. Three different methods were used to separate the nuclear membrane from whole purified nucleus. Nuclear compartment markers such as anti-Histone3 and anti-Fibrillarin was used in this step. In the study, Inner Nuclear Membrane protein (AtSUN2) was used as a nuclear membrane marker. All things considered, we conclude that the method with DNase digestion and high centrifugation speed (first method ) is a more effective way in separation of nuclear membrane fractions because the low centrifugal speed (second method) does not appear to be sufficient for separating the nuclear membrane, and the third method does not seem to be a very effective way as it requires a high centrifugal speed.

The intramembrane protease SPP impacts morphology of the endoplasmic reticulum by triggering degradation of morphogenic proteins

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway.

Quantitative Lipid Droplet Proteomics Reveals Mycobacterium tuberculosis Induced Alterations in Macrophage Response to Infection.

Growing evidence suggests the importance of lipid metabolism in pathogenesis of tuberculosis. Neutral lipids form the majority of lipids in a caseous granuloma, a pathology characteristic of tuberculosis. Cytosolic lipid droplets (LDs) of macrophages form the store house of these lipids and have been demonstrated to contribute to the inflammatory response to infection. The proteome of lipid droplets reflects the mechanisms of lipid metabolism active under a condition. However, infection induced changes in the proteome of these dynamic organelles remains elusive. Here, we employed quantitative proteomics to identify alterations induced upon infection with live Mycobacterium tuberculosis (Mtb) in comparison with heat killed bacilli or uninfected macrophages. We found increased abundance of proteins coupled with lipid metabolism, protein synthesis, and vesicular transport function in LDs upon infection with live Mtb. Using biochemical methods and microscopy, we validated ADP-ribosyltransferase (Arf)-like 8 (ARL8B) to be increased on the lipid droplet surface of live Mtb infected macrophages and that ARL8B is a bonafide LD protein. This study provides the first proteomic evidence that the dynamic responses to infection also encompass changes at the level of LDs. This information will be important in understanding how Mtb manipulates lipid metabolism and defense mechanisms of the host macrophage.

Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT.

The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome.

Proteomic analysis of insulin secretory granules in INS-1\xa0cells by protein correlation profiling

Insulin secretory granules (ISGs), a group of distinguishing organelles in pancreatic β cells, are responsible for the storage and secretion of insulin to maintain blood glucose homeostasis. The molecular mechanisms of ISG biogenesis, maturation, transportation, and exocytosis are still largely unknown because the proteins involved in these distinct steps have not been fully identified. Subcellular fractionation by density gradient centrifugation has been successfully employed to analyze the proteomes of numerous organelles. However, use of this method to elucidate the ISG proteome is limited by co-fractionated contaminants because ISGs are very dynamic and have abundant exchanges or contacts with other organelles, such as the Golgi apparatus, lysosomes, and endosomes. In this study, we developed a new strategy for identifying ISG proteins by protein correlation profiling (PCP)-based proteomics, which included ISG purification by OptiPrep density gradient centrifugation, label-free quantitative proteome, and identification of ISG proteins by correlating fractionation profiles between candidates and known ISG markers. Using this approach, we were able to identify 81 ISG proteins. Among them, TM9SF3, a nine-transmembrane protein, was considered a high confidence ISG candidate protein highlighted in the PCP network. Further biochemical and immunofluorescence assays indicated that TM9SF3 localized in ISGs, suggesting that it is a potential new ISG marker.Graphical abstract

Effect of acupuncture on hippocampal mitochondrial proteome expression in SAMP8 mouse model with Alzheimer disease

To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone 8 (SAMP8) mice models with Alzheimer disease (AD), and to explore the possible protective mechanism of acupuncture on mitochondria. Sixty 6-month-old male SAMP8 mice were randomly divided into an acupuncture at acupoint group, an acupuncture at non-acupoint group and a model group, 20 mice in each group. The 20 male senescence-accelerated mouse/resistance 1 (SAMR1) mice of the same age were used as a normal control group. Shenshu (BL 23), Baihui (GV 20), Xuehai (SP 10) and Geshu (BL 17) were selected for acupuncture intervention in acupuncture at acupoint group. After an 8-week intervention, mitochondrial tissues were extracted from the hippocampus. Differentially expressed proteins were identified by subcellular organelle proteomics. Western blot was used to verify the expressions of some related proteins in hippocampal mitochondria. Compared with the model group, there were 13 differentially expressed protein spots in the acupuncture at acupoint group, of which, 9 were up-regulated, including neurofilament light polypeptide (NFL), actin (cytoplasmic 1, database ID: ACTB), tubulin beta-2A chain (TBB2A), tropomodulin-2 (TMOD2), pyruvate dehydrogenase E1 component subunit beta (PDHE1-β), NADH-ubiquinone oxidoreductase 75 kDa subunit (database ID: NDUS1), heat shock cognate 71 kDa protein (HSC71), pyruvate dehydrogenase E1 component subunit alpha (PDHE1-α) and ATP synthase beta subunit (ATP-β); 4 were down-regulated, including glial fibrillary acidic protein (GFAP), pyruvate dehydrogenase phosphatase 1 (PDP1), mitochondrial-processing peptidase subunit alpha (MMP-α) and adenosine kinase (ADK). According to the information provided in the protein database, most of the differentially expressed proteins involve the regulation of mitochondrial function and structure. The expression levels of NFL and TBB2A in the normal control group and the acupuncture at acupoint group were significantly higher than those in the acupuncture at non-acupoint group (P 0.05). Acupuncture may achieve the potential therapeutic effect on AD by regulating the structure and functional proteins of hippocampal mitochondria.

The Craft of Peroxisome Purification-A Technical Survey Through the Decades.

Purification technologies are one of the working horses in organelle proteomics studies as they guarantee the separation of organelle-specific proteins from the background contamination by other subcellular compartments. The development of methods for the separation of organelles was a major prerequisite for the initial detection and characterization of peroxisome as a discrete entity of the cell. Since then, isolated peroxisomes fractions have been used in numerous studies in order to characterize organelle-specific enzyme functions, to allocate the peroxisome-specific proteome or to unravel the organellar membrane composition. This review will give an overview of the fractionation methods used for the isolation of peroxisomes from animals, plants and fungi. In addition to "classic" centrifugation-based isolation methods, relying on the different densities of individual organelles, the review will also summarize work on alternative technologies like free-flow-electrophoresis or flow field fractionation which are based on distinct physicochemical parameters. A final chapter will further describe how different separation methods and quantitative mass spectrometry have been used in proteomics studies to assign the proteome of PO.

Alteration of Liver Peroxisomal and Mitochondrial Functionality in the NZO Mouse Model of Metabolic Syndrome.

Metabolic syndrome (MetS) consists of five risk factors: elevated blood pressure and fasting glucose, visceral obesity, dyslipidemia, and hypercholesterinemia. The physiological impact of lipid metabolism indicated as visceral obesity and hepatic lipid accumulation on MetS is still under debate. One major cause of disturbed lipid metabolism might be dysfunction of cellular organelles controlling energy homeostasis, i.e., mitochondria and peroxisomes. The New Zealand Obese (NZO) mouse model exhibits a polygenic syndrome of obesity, insulin resistance, triglyceridemia, and hypercholesterolemia that resembles human metabolic syndrome. We applied a multi-omics approach combining lipidomics with liver transcriptomics and top-down MS based organelle proteomics (2D-DIGE) of highly enriched mitochondria and peroxisomes in male mice, to investigate molecular mechanisms related to the impact of lipid metabolism in the pathophysiology of the metabolic syndrome. Proteome analyses of liver organelles indicate differences in fatty acid and cholesterol metabolism, mainly influenced by PG-C1α/PPARα and other nuclear receptor mediated pathways. These results are in accordance with altered serum lipid profiles and elevated organelle functionality. These data emphasize that metabolic syndrome is accompanied with increased mitochondria and peroxisomal activity to cope with dyslipidemia and hypercholesterinemia driven hepatic lipid overflow in developing a fatty liver.

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.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.