Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Membrane dynamics and the biogenesis of lysosomes

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Compartmentalization of the Edinburgh Human Metabolic Network

Background  

Direct in vivo investigation of human metabolism is complicated by the distinct metabolic functions of various sub-cellular organelles. Diverse micro-environments in different organelles may lead to distinct functions of the same protein and the use of different enzymes for the same metabolic reaction. To better understand the complexity in the human metabolism, a compartmentalized human metabolic network with integrated sub-cellular location information is required.     

Improved sub-cellular resolution via simultaneous analysis of organelle proteomics data across varied experimental conditions

Spatial organisation of proteins according to their function plays an important role in the specificity of their molecular interactions. Emerging proteomics methods seek to assign proteins to sub-cellular locations by partial separation of organelles and computational analysis of protein abundance distributions among partially separated fractions. Such methods permit simultaneous analysis of unpurified organelles and promise proteome-wide localisation in scenarios wherein perturbation may prompt dynamic re-distribution. Resolving organelles that display similar behavior during a protocol designed to provide partial enrichment represents a possible shortcoming. We employ the Localisation of Organelle Proteins by Isotope Tagging (LOPIT) organelle proteomics platform to demonstrate that combining information from distinct separations of the same material can improve organelle resolution and assignment of proteins to sub-cellular locations. Two previously published experiments, whose distinct gradients are alone unable to fully resolve six known protein-organelle groupings, are subjected to a rigorous analysis to assess protein-organelle association via a contemporary pattern recognition algorithm. Upon straightforward combination of single-gradient data, we observe significant improvement in protein-organelle association via both a non-linear support vector machine algorithm and partial least-squares discriminant analysis. The outcome yields suggestions for further improvements to present organelle proteomics platforms, and a robust analytical methodology via which to associate proteins with sub-cellular organelles.     

Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.     

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.     

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.     

Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage-fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.     

Vps39 is required for ethanolamine-stimulated elevation in mitochondrial phosphatidylethanolamine

Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a ‘moonlighting’ role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ, implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria.     

GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.     

Peroxisome homeostasis in Hansenula polymorpha

Peroxisomes are essential organelles in many eukaryotes. Until recently, the main focus of the investigations concerning these important organelles was to understand the biogenesis of the peroxisome (induction, proliferation and matrix protein import). However, when peroxisomes become redundant they are quickly degraded by highly selective processes known as pexophagy. The first molecular studies on pexophagy have indicated that this process shares many features with certain transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole targeting and endocytosis). Nevertheless, recent data demonstrate that in addition to common genes also unique genes are required for these transport processes. The main focus for the future should therefore be on identifying the unique determinants of pexophagy. Earlier results suggest that in the methylotrophic yeast Hansenula polymorpha proteins located on the peroxisome itself are required for pexophagy. Thus, it has become essential to study in detail the role of peroxisomal membrane proteins in the degradation process. This review highlights the main achievements of the last few years, with emphasis on H. polymorpha.     

Viral Bimolecular Fluorescence Complementation: A Novel Tool to Study Intracellular Vesicular Trafficking Pathways

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.     

A non-aqueous method for the sub-cellular fractionation of cotyledons from dormant seeds of Phaseolus vulgaris L.

A method is described for the sub-cellular ractionation of cotyledons from non-germinated Phaseolus vulgaris seeds in non-aqueous density gradients of potassium iodide in glycerol. The major organelles (protein bodies, cell walls and starch grains) are well resolved as adjudged by morphology and protein subunit patterns of gradient zones and are remarkably stable if water is rigorously excluded. The flexibility of the system is exploited in establishing the cytoplasmic location of the oligosaccharide fraction and in the large-scale preparative isolation of the sub-cellular components. The composition and morphology of the sub-cellular components are described and discussed.Abbreviation BAPNA are is defined as hydrolytic activity directed towards the substrate -N-benzoyl-L-arginine-p-nitroanilide     

Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography

Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.     

Transport of proteins in eukaryotic cells: more questions ahead

Some newly synthesized proteins contain signals that direct their transport to their final location within or outside of the cell. Targeting signals are recognized by specific protein receptors located either in the cytoplasm or in the membrane of the target organelle. Specific membrane protein complexes are involved in insertion and translocation of polypeptides across the membranes. Often, additional targeting signals are required for a polypeptide to be further transported to its site of function. In this review, we will describe the trafficking of proteins to various cellular organelles (nucleus, chloroplasts, mitochondria, peroxisomes) with emphasis on transport to and through the secretory pathway.     

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta-subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.     

Proteomic analysis of B-cell malignancies

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.     

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.     

Organelle identity and the targeting of peripheral membrane proteins

Within the secretory pathway, most proteins involved in vesicle formation, motor recruitment and vesicle tethering are not integral membrane proteins but, rather, peripheral membrane proteins recruited to the relevant organelles from the cytosol. From recent studies on diverse organelles, it appears that such recruitment is usually mediated by binding to a labile determinant, such as an activated G protein or a short-lived lipid species, whose distribution is restricted to a single organelle. This suggests that these determinants are what specify organelle identity, and raises interesting questions about how they are generated in an organelle-specific fashion.