Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

Localization of organelle proteins by isotope tagging (LOPIT)

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.     

Mapping the subcellular protein distribution in three human cell lines

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of more than 3500 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together.     

Subcellular fractionation methods and strategies for proteomics

Developments in subcellular fractionation strategies have provided the means to profile and analyze the protein composition of organelles and cellular structures by proteomics. Here, we review the application of classical (e.g. density gradient centrifugation) and emerging sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and statistical/bioinformatics tools for the prediction of protein localization in subcellular proteomics. We also review the validation methods currently used (such as microscopy, RNA interference and multiple reaction monitoring) and discuss the importance of verification of the results obtained in subcellular proteomics. Finally, the numerous challenges facing subcellular proteomics including the dynamics of organelles are being examined. However, complementary approaches such as modern statistics, bioinformatics and large-scale integrative analysis are beginning to emerge as powerful tools to proteomics for analyzing subcellular organelles and structures.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Integrative analysis of the mitochondrial proteome in yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.     

Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques

High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein gylcosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.     

Methods of quantitative proteomics and their application to plant organelle characterization

Many cell biologists wish to know the subcellular localization of proteins of interest. Proteomics methods have the potential to describe the entire protein content of organelles. However, practical limitations in organelle isolation and analysis of low abundance proteins have meant that organelle proteomics has had, until recently, only limited success. Some examples of quantitative proteomic methods and their use in the study of plant organelle proteomes are discussed here. It is concluded that 2D-difference gel electrophoresis (2D-DIGE) as well as differential isotope tagging strategies coupled to non-gel-based LC-MS are proving useful in this area of research.     

The Organelle Proteome of the DT40 Lymphocyte Cell Line

A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and endocytic pathway a high proportion of proteins are not uniquely localized to a single organelle, emphasizing the dynamic steady-state nature of intracellular compartments in eukaryotic cells.The chicken pre-B cell line DT40 exhibits a remarkably high ratio of targeted to random integration for transfected DNA constructs. This property is unusual in vertebrate cell lines and enables targeted gene disruption experiments to be carried out with relative ease (1). Consequently, DT40 has become a major research tool for the molecular dissection of a wide range of cellular and biochemical mechanisms in a vertebrate context, including membrane traffic, signal transduction, and cell cycle (2).Proteins in eukaryotic cells are organized according to their functions within a dynamic network of membranes. Localization is therefore paramount in assigning functions to uncharacterized proteins and understanding the processes occurring in subcellular compartments. An increased knowledge of the protein localization within the DT40 cell line would be of great value. Traditional localization methods such as immunofluorescence microscopy are typically low throughput and are more suitably applied to the study of specific proteins of interest rather than the cataloguing of large numbers of proteins. Recent developments in proteomics have made it possible to analyze the protein composition of organelles using a variety of different approaches. Several groups have utilized label-free quantitative proteomics in the high throughput assignment of proteins to subcellular compartments. In one approach, protein correlation profiling, proteins from enriched organelle fractions are quantified by peptide ion intensity measurements (3, 4). Other similar methods employ quantitation by spectral counting, recording the number of ions detected per protein (5, 6). Localization of organelle proteins by isotope tagging (LOPIT)¹ is a complementary approach, which employs isotope labeling for quantitation (7–9). Rather than processing each sample separately as in label-free techniques, differentially labeled fractions are pooled early in the LOPIT protocol. This has the important advantage of reducing the points at which variation might be introduced into the data.LOPIT begins with the partial separation of organelles by density gradient centrifugation and relies on the assumption that proteins from each organelle co-fractionate. Protein profiles along the gradient are quantified by the use of isotopically coded tags in conjunction with two-dimensional liquid chromatography of peptides and tandem mass spectrometry. Multivariate statistical techniques are then used to assign localizations to proteins by comparing their gradient profiles to those of established organelle markers in an unbiased manner. The major strength of such an approach is that it enables residents of different subcellular compartments to be resolved even if their gradient distributions overlap, and genuine organelle constituents can be readily distinguished from contaminants.Here we use LOPIT to produce the first proteomic analysis of the major organelles of DT40. We have reproducibly identified 1090 proteins through the parallel analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins. We use the distributions of 102 known organelle resident proteins as a basis to assign a further 223 proteins to five organelles: 79 to the endoplasmic reticulum (ER), 42 to the Golgi, 2 to the lysosome, 31 to the mitochondrion, and 69 to the plasma membrane (PM). We also demonstrate the resolution of components of the vesicular transport machinery. A striking finding is that a high proportion of identified proteins are not localized to a single organelle. This indicates that at steady state a substantial fraction of proteins are in transit between compartments, emphasizing the dynamic nature of intracellular organelles in eukaryotic cells. Our results represent the first application of LOPIT to a vertebrate system, provide the first organelle proteomic analysis of any lymphocyte cell line, and establish a major resource for the DT40 community.     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.     

A single fixation protocol for proteome-wide immunofluorescence localization studies

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.     

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress.     

Global organellar proteomics

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

The proteome of lysosomes

Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.     

Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans

Nematode sperm contain unusual organelles, membranous organelles, which undergo dramatic morphological changes during spermatogenesis. Early in spermatogenesis, the membranous organelle functions to transport sperm specific components to the spermatids; later, during the formation of the crawling spermatozoa, it adds new components to the cell surface as it fuses with the plasma membrane. Genetic analysis of spermatogenesis in the nematode Caenorhabditis elegans has revealed mutations that specifically disrupt the proper cellular localization and morphogenesis of this organelle. In animals homozygous for the either the known deficiency hcDf1 or the probable deficiency h12, the membranes of the membranous organelles are aberrantly covered with ribosomes. A mutation in the spermatogenesis-defective spe-10 gene causes severe defects in the morphogenesis of a fibrous body-membranous organelle complex. In both cases, these mutations also disrupt the proper localization of both nuclei and membranous organelles in haploid spermatids and spermatozoa.