Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction,Molecular Transport,and Callose Biosynthesis

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.The plasma membrane (PM)¹ is considered as one of the most interactive and dynamic supramolecular structures of the cell (1, 2). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4–8). Typically, PM proteins are either embedded in the phospholipid bilayer through transmembrane helices or less tightly bound to the membrane through reversible or irreversible surface interactions. In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9, 10). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9, 10). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9–11). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10, 11). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM.Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13–15), Arabidopsis (16), barrel clover (Medicago truncatula) (17), rice (18), oat, and rye (19). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15–19). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15–19). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells.Although several reports describe the partial characterization of DRMs from higher plants (13–23), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins.     

Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.Phagocytosis, the mechanism by which large particles are internalized, leads to the formation of phagosomes, a specialized organelle in which the engulfed material is degraded (1, 2). In mammals, various cells including macrophages, neutrophils and dendritic cells display remarkable phagocytic activities, rapidly eliminating microorganisms, foreign inert particles, and apoptotic cells. The killing of microorganisms by professional phagocytes precludes the emergence of infectious diseases. This innate immune process is followed by the degradation of microbes in a highly concentrated mixture of hydrolases, activated by the acidic pH generated in the phagosome lumen, generating antigenic peptides that are displayed at the cell surface, enabling their recognition by T lymphocytes (3). The peptides not loaded on MHC molecules are fully degraded in phagolysosomes and the end products are likely recycled from phagosomes by a variety of transporters (1). The establishment of these functional properties involves a complex remodeling of phagosomes, referred to as phagolysosome biogenesis (4, 5). This highly regulated process requires the fusion of nascent phagosomes with trans Golgi-derived vesicles, early endosomes, late endosomes and ultimately lysosomes (1, 2). These fusion events are believed to alter significantly the proteome of phagosomes during phagolysosome biogenesis and regulate their functional properties (6).The capacity to kill and degrade microbes is one of the many functions that phagosomes acquire during phagolysosome biogenesis. In a previous study, we identified more than 140 proteins associated with phagosomes (7), leading to the proposal of novel mechanisms to explain phagosomal functions such as antigen cross-presentation (8). This proteomics study also shown the presence on phagosomes of proteins known to segregate into lipid rafts at the cell surface, such as flotillin-1 and prohibitin, leading to the proposal that membrane microdomains might also assemble on phagosomes. At the plasma membrane, these structures constitute foci of specialized functions, notably for signal transduction (9). Further biochemical and morphological analyses confirmed the presence of membrane microdomains on phagosomes (10). The role of membrane microdomains and the molecular nature of these structures in phagosomes is still poorly understood. Recent data indicated that two phagosomal protein complexes, V-ATPase and NADPH oxidase may use membrane microdomains as assembly platforms (11). Furthermore, the potential involvement of phagosome microdomains in innate immunity was highlighted by the finding that at least two unrelated pathogens, the Gram-negative bacteria Brucella and the intracellular parasite Leishmania donovani, target phagosome lipid rafts as a strategy to evade host-defense mechanisms (12–14). Hence, the molecular characterization of the detergent-soluble and -insoluble fractions isolated from phagosomes should provide unique insights into the mechanisms used by pathogens to alter the functional properties of this organelle. Different approaches have been used to study membrane microdomains, including imaging techniques such as fluorescence resonance energy transfer, fluorescence photoactivation localization microscopy, as well as cell fractionation procedures using non-ionic detergents to enrich detergent-resistant membrane domains (15). Imaging approaches highlighted the fact that cholesterol-enriched membrane microdomains are dynamic microscopic structures of less than 20 nm in range. On the other hand, detergent-based fractionation approaches have been extensively used to identify key components of membrane microdomains, including series of signaling factors (16–18). Although the exact nature and the level of correspondence of the membrane microdomains studied by the morphological and biochemical approaches is still actively debated, similar sets of proteins have been identified in these structures (15).In the present study we used quantitative proteomics approach to characterize, for the first time, the modifications of lipid rafts proteins occurring during the biogenesis of an intracellular organelle. Our data indicate that segregation of sets of proteins in sub-regions of the phagosome membrane occurs throughout the biogenesis and maturation of phagolysosome, introducing the concept that spatiotemporal reorganization of the phagosome proteome plays a key role in the establishment of the functional properties of this organelle.     

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.The plasma membrane incorporates a broad spectrum of proteins covering mainly different structural, signaling or transport functionalities. Being the first semipermeable cell barrier to its surrounding environment the plasma membrane is important for metabolite transport as well as initiation point of several signaling processes (1–4). To maintain cell homeostasis, protein activity as well as complex formation through protein protein interactions (PPI) need to be tightly regulated. The major regulating mechanisms are postranslational modification of proteins and modulated abundances of proteins present in the plasma membrane. Another potential regulating mechanism became apparent with the discovery of sterol and sphingolipid enriched domains (microdomains) in the plasma membrane (5–8, 3). Microdomain like structures have been shown to form spontaneously in artificial plasma membranes (9). After a decade of research on these structures, microdomains turned out to be particularly involved in signaling and transport processes incorporating a specific set of proteins. Microdomains provide subcompartments in the plasma membrane with specific physicochemical properties that on specific sterol protein interactions might alter protein activity or PPIs. With the discovery of microdomains the fluid lipid mosaic model was extended by distinguishing two plasma membrane phases, an ordered phase of lower density (L_o phase) enriched in sterols, sphingolipids and long chain fatty acids and a disordered phase of higher density (L_d phase). From isolated plasma membranes a lower density and a higher density membrane fraction can be separated in a sucrose gradient after treatment with non-ionic detergents. The resulting detergent resistant membrane fraction (DRM)¹ is related to L_o phase and high density detergent soluble membrane fraction (DSF) relates to L_o phases. Although it is still under debate how well DRMs represent native plasma membrane microdomains (10–12), research on protein-sterol interactions is possible by usage of sterol depleting agents like methyl-β-cyclodextrin mβcd (13). Therefore mβcd is suitable for detecting false positive cholesterol protein interactions in DRM studies (14–19). Proteins depleted on mβcd treatment are finally considered to be sterol dependent (15–17). To compare the mβcd treatment for disturbing the sterol distribution in the L_o fraction, we studied the sterol biosynthesis deficient mutant smt1. (20) smt1 carries a point mutation in the smt1 locus, encoding the sterol methyltransferase 1 and it exhibits a dwarf-like phenotype on whole plant level (20). In total, three sterol methyl transferases are encoded in Arabidopsis where SMT1 catalyzes the first step in the sterol biosynthesis by adding a methyl group at C24 of the sterol precursor cycloartenol. SMT2 and SMT3 act at later steps and were shown to be functionally redundant as C-24 sterol methyltransferases at the branching in sterol synthesis that either leads to sitosterol or campesterol (21). The total sterol composition in smt1 mutants was shown to be different from wild type, with the major phytosterols like sitosterol, stigmasterol, and brassicasterol being strongly depleted. In contrast, other sterol species remained unaltered and some even increased (20, 21). So far, it remains unclear how the altered sterol-composition of the smt1 mutant affects sterol-protein interactions. In this study, using the newly developed algorithm Unicorn, we compared changes in protein distributions between DRM and DSF after biochemical mβcd treatment and on endogenous alterations in sterol composition in smt1 to improve understanding of sterol–protein interactions.     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.Despite the importance of cell surface biology, the conventional shotgun proteomics strategy generally underrepresents the membrane proteome because of inadequate solubilization and protease digestion (1, 2). The ageless gel strategy, consisting of SDS-PAGE followed by in-gel digestion, can partially solve this problem (3–5), but the recovery from in-gel digestion is generally lower than that from in-solution digestion, and this approach is far from suitable for a rapid, simple, and high throughput automated system. Numerous approaches have been reported to overcome the difficulties in membrane proteome analysis, such as the use of surfactants (2, 6–11), organic solvents (6, 7, 12–15), or chaotropic reagents (2, 6, 16). Acid-labile surfactants, such as RapiGest SF, are among the most promising additives to enhance protein solubilization without interfering with LC-MS performance (6, 10, 17–19). However, the cleavage step at acidic pH causes loss of hydrophobic peptides because of coprecipitation with the hydrophobic part of RapiGest SF (20). Recently, we developed a new protocol to dissolve and digest membrane proteins with the aid of a removable phase transfer surfactant (PTS),1 such as sodium deoxycholate (SDC) (20). The solubility of membrane proteins with SDC was comparable to that with sodium dodecyl sulfate. In addition, the activity of trypsin was enhanced ∼5-fold in the presence of 1% SDC because this rapid PTS method mimics conditions in the alimentary canal in which bile salts such as cholate and deoxycholate are secreted together with trypsin. After tryptic digestion, SDC is removed prior to LC-MS/MS analysis by adding an organic solvent followed by pH-induced transfer of the surfactant to the organic phase, whereas tryptic peptides remain in the aqueous phase. This protocol offers a significant improvement in identifying membrane proteins by increasing the recovery of hydrophobic tryptic peptides compared with the protocols using urea and RapiGest SF.The goal of this study is to establish a membrane proteomics method that is unbiased with respect to protein solubility, hydrophobicity, and protein abundance; i.e. membrane proteins can be as efficiently extracted and digested as soluble proteins. So far, to our knowledge, little information about the recovery of the membrane proteome has been reported. Instead, the number of identified membrane proteins or the content of membrane proteins identified in the membrane-enriched fraction has been used as an indicator of the efficiency of procedures for membrane proteome analysis (4, 5, 21–23). However, these parameters usually depend on the experimental conditions, including the sample preparation procedure and LC-MS instrument used. Therefore, it is difficult to compare data obtained with these protocols except in the case of direct comparison. Furthermore, there has been no report quantitatively comparing the recovery of membrane proteome with that of soluble proteins.In this study, we used a modified version of our PTS protocol with immobilized trypsin columns to reduce the digestion time and evaluated its suitability for unbiased quantitation of the membrane proteome. In addition, we applied this protocol to estimate the copy numbers per cell of 1,453 proteins, including 545 membrane proteins, using the exponentially modified protein abundance index (emPAI). Finally, this rapid and unbiased PTS protocol was applied to the quantitative analysis of Escherichia coli BW25113 wild-type and relA knock-out (KO) strains.     

Quantitative Proteomics Reveals a Dynamic Association of Proteins to Detergent-resistant Membranes upon Elicitor Signaling in Tobacco

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used ¹⁴N/¹⁵N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.The plasma membrane of eukaryotes delineates the interface between the cell and the environment. Thus it is particularly involved in environmental signal recognition and their transduction into intracellular responses, playing a crucial role in many essential functions such as cell nutrition (involving transport of solutes in and out of the cell) or response to environmental modifications (including defense against pathogens).Over the last 10 years, a new aspect of the plasma membrane organization has arisen from biophysical and biochemical studies performed with animal cells. Evidence has been given that the various types of lipids forming this membrane are not uniformly distributed inside the bilayer but rather spatially organized (1). This leads in particular to the formation of specialized phase domains, also called lipid rafts (2, 3). Recently a consensus emerged on the characteristics of these domains. Both proteins and lipids contribute to the formation and the stability of membrane domains that should be called “membrane rafts” and are envisaged as small (10–200-nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes (4). Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (5). Because of their particular lipidic composition (enrichment in sterol, sphingolipids, and saturated fatty acids), these domains form a liquid ordered phase inside the membrane. This structural characteristic renders them resistant to solubilization by non-ionic detergents, and this property has been widely used to isolate lipid rafts as detergent-resistant membranes (DRMs)¹ for further analysis (1). The most important hypothesis to explain the function of these domains is that they provide for lateral compartmentalization of membrane proteins and thereby create a dynamic scaffold to organize certain cellular processes (5). This ability to temporally and spatially organize protein complexes while excluding others conceivably allows for efficiency and specificity of cellular responses. In yeasts and animal cells, the association of particular proteins with these specialized microdomains has emerged as an important regulator of crucial physiological processes such as signal transduction, polarized secretion, cytoskeletal organization, generation of cell polarity, and entry of infectious organisms in living cells (6). Much of the early evidence for a functional role of lipid rafts came from studies of hematopoietic cells in which multichain immune receptors including the high affinity IgE receptor (FcεRI), the T cell receptor, and the B cell receptor (BCR) translocate to lipid rafts upon cross-linking (7). Moreover this signaling involves the relocalization of several proteins; for instance the ligation of the B cell antigen receptor with antigen induced a dissociation of the adaptor protein ezrin from lipid rafts (8). This release of ezrin acts as a critical trigger that regulates lipid raft dynamics during BCR signaling.In plants, the investigations of the presence of such microdomains are very recent and limited to a reduced number of publications (for a review, see Ref. 9). A few years ago, Peskan et al. (10) reported for the first time the isolation of Triton X-100-insoluble fractions from tobacco plasma membrane. Mongrand et al. (11) provided a detailed analysis of the lipidic composition of such a detergent-resistant fraction indicating that it was highly enriched in a particular species of sphingolipid (glycosylceramide) and in several phytosterols (stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol) compared with the whole plasma membrane from which it originates. Similar results were then obtained with DRMs prepared from Arabidopsis thaliana cell cultures (12) and from Medicago truncatula roots (13). So the presence in plant plasma membrane of domains sharing with animal rafts a particular lipidic composition, namely strong enrichment in sphingolipids together with free sterols and sterol conjugates, the latter being specific to the plant kingdom (11, 13), now seems established. In plant only a few evidences suggest in vivo the role of dynamic clustering of plasma membrane proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the plasma membrane (PM), a process reminiscent of lipid rafts (14). Consistently a proteomics study of tobacco DRMs led to the identification of 145 proteins among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (15). This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in such physiological functions.Cryptogein belongs to a family of low molecular weight proteins secreted by many species of the oomycete Phytophthora named elicitins that induce a hypersensitivity-like response and an acquired resistance in tobacco (16). To understand molecular processes triggered by cryptogein, its effects on tobacco cell suspensions have been studied for several years. Early events following cryptogein treatment include fixation of a sterol molecule (17, 18); binding of the elicitor to a high affinity site located on the plasma membrane (19); alkalinization of the extracellular medium (20); efflux of potassium, chloride, and nitrate (20, 21); fast influx of calcium (22); mitogen-activated protein kinases activation (23, 24); nitric oxide production (25, 26); and development of an oxidative burst (27, 28). We previously identified NtrbohD, an NADPH oxidase located on the plasma membrane, as responsible for the reactive oxygen species (ROS) production occurring a few minutes after challenging tobacco Bright Yellow 2 (BY-2) cells with cryptogein (29). The fact that most of these very early events involve proteins located on the plasma membrane and that one of them, NtrbohD, has been demonstrated as exclusively associated to DRMs in a sterol-dependent manner (30) prompted us to analyze the modifications of DRM proteome after cryptogein treatment. In the present study, we aimed to confirm the hypothesis that, as observed in animal cells, the dynamic association to or exclusion of proteins from lipid rafts could participate in the signaling process occurring during biotic stress in plants.To achieve this goal, we had to set up a quantitative assay allowing a precise comparison of the amounts of each protein in DRMs extracted from either control or cells treated with cryptogein. Among several technologies, we excluded DIGE (31), recently used to analyze whole cell proteome variations in plants (32–34), because membrane proteins are poorly soluble in the detergents used for two-dimensional electrophoresis; this limitation is all the more marked for proteins selected on the basis of their insolubility in non-ionic detergent, the criteria for DRMs isolation. Stable isotope labeling of proteins or peptides combined with MS analysis represents alternative strategies for accurate, relative quantification of proteins on a global scale (35, 36). In these approaches, proteins or peptides of two different samples are differentially labeled with stable isotopes, combined in an equal ratio, and then jointly processed for subsequent MS analysis. Relative quantification of proteins is based on the comparison of signal intensities or peak areas of isotope-coded peptide pairs extracted from the respective mass spectra. Stable isotopes can be introduced either chemically into proteins/peptides via derivatization of distinct functional groups of amino acids or metabolically during protein biosynthesis (for a review, see Ref. 37). Metabolic labeling strategies are based on the in vivo incorporation of stable isotopes during growth of organisms. Nutrients or amino acids in a defined medium are replaced by their isotopically labeled (¹⁵N, ¹³C, or ²H) counterparts eventually resulting in uniform labeling of proteins during the processes of cell growth and protein turnover (38). As a consequence, differentially labeled cells or organisms can be combined directly after harvesting. This minimizes experimental variations due to separate sample handling and thus allows a relative protein quantification of high accuracy.¹⁴N/¹⁵N labeling has been recently proved to be suitable for comparative experiments performed with whole plants (39–42) and in plant suspension cells where the level of incorporation is equal to the isotopic purity of the salt precursor (43, 44). It has been used successfully to analyze some variations induced in A. thaliana plasma membrane proteome following heat shock (45) or cadmium exposure (46) and to compare phosphorylation levels of plasma membrane proteins after challenge of Arabidopsis cells with elicitors of defense reaction (47). In the present study, we used a mineral ¹⁴N/¹⁵N metabolic labeling of tobacco BY-2 cells before treatment with cryptogein and subsequent isolation of DRMs. The DRM proteins were further analyzed by one-dimensional SDS-PAGE and digested by trypsin, and peptides were subjected to microcapillary high performance LC-MS/MS (nano-LC-MS/MS). This metabolic method allowed a complete labeling of the proteome, and consequently a major drawback of this method is probably the difficulty to perform an exhaustive analysis of the very large amount of data generated. To solve this problem, a new quantification module of the MFPaQ software (48) was developed, allowing the automatic quantification of the identified peptides. The results derived from the program were validated through a comparison with manual quantification. Thus, we achieved the complete analysis of the DRM proteome variation and identified four proteins whose abundance in DRMs was decreased and one that was enriched in DRMs upon elicitation. The biological relevance of these results, which indicate that, in plant as in animals, the dynamic association of proteins to membrane domains is part of a signaling pathway, will be further discussed.     

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD)¹ is severe synaptic loss (1–3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10–12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13–18). Post-translational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC–tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and age-matched controls, as well as a study of SNARE complex protein levels in brain tissue from prion-infected mice.     

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking.To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer''s clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.Plasmodium falciparum, the most deadly agent of human malaria, caused around 216 million infections and 655,000 deaths in 2010. The complex parasite life cycle involves the development in a mosquito vector of the Anopheles genus and eventual migration to a human host. In this host, asymptomatic multiplication in the liver cells is followed by parasite release into the bloodstream and erythrocyte invasion. Inside the erythrocytes, parasites grow (trophozoite stage) and multiply asexually (schizont stage), developing into highly specialized invasive forms (merozoites). A fraction of parasites differentiate into gametocytes, the gamete precursors necessary to complete the transmission cycle. Parasite blood stages, responsible for malaria pathogenesis and transmission, actively remodel the host erythrocyte, generating novel membrane compartments to sustain the export and sorting of proteins to the host cell cytosol, membrane skeleton, and plasma membrane. The parasitophorous vacuole membrane (PVM),¹ which surrounds the parasite throughout the erythrocytic cycle, is the site where exported proteins are translocated into the erythrocyte cytosol (1, 2). Membrane-bound structures of parasite origin, the so-called Maurer''s clefts (MCs) (3, 4), form functionally independent compartments at the red blood cell (RBC) periphery and mediate the sorting/assembly of virulence factors en route to the host cell surface (5). In addition, populations of different vesicles (25 and 80 nm) were identified in the RBC cytosol, suggesting the presence of vesicular mediated trafficking for the delivery of cargo to different destinations (6).Membranes are important sites for cellular signaling events, and many proteins with therapeutic potential localize in these cellular compartments (7, 8). Membrane microdomains enriched in sphingolipids and cholesterol, also referred to as lipid rafts, have been extensively studied in different cell types and gained particular interest for their roles in infection and pathogenesis (8, 9). These assemblies are small and dynamic and can be stabilized to form larger microdomains implicated in a wide range of fundamental cellular processes, which vary depending on cell type (10). Sphingolipids exhibit strong lateral cohesion, generating tightly packed regions in the membrane bilayer, and cholesterol acts as a spacer present in both membrane leaflets generating stable, liquid-ordered phase domains in the membrane bilayer (11). Distinct biochemical properties render these membrane assemblies insoluble in nonionic detergents at low temperature, allowing for their enrichment as detergent-resistant membranes (DRMs). Proteins with DRM-raft affinity include glycosylphosphatidyl inositol (GPI)-anchored proteins and acylated, myristoylated, and palmitoylated proteins (11). DRM rafts also restrict free diffusion of membrane proteins, thereby directing the trafficking of proteins and lipids to and from cellular compartments. Because of their endocytic and receptor clustering capacity, an increasing number of pathogens, including Plasmodium falciparum, utilize them when interacting with their target cells for invasion (9, 12).Even though cholesterol-rich membrane microdomains are implicated in fundamental processes in the parasite life cycle, Plasmodium is unable to synthesize sterols and depends entirely on hosts for its cholesterol supply. During merozoite invasion, lipid and protein components of the erythrocyte rafts are selectively recruited and incorporated into the nascent PVM (13, 14). Plasmodium liver stages utilize cholesterol internalized by low-density lipoprotein and synthesized by hepatocytes (15).To shed light on the organization and dynamics of these assemblies during parasite development inside the infected cell, we identified and validated the DRM-raft proteome of the P. falciparum trophozoite/early schizont. Detected proteins only partially overlap with DRM components of the P. falciparum late schizonts (16, 17) or the mixed blood stages of the rodent malaria agent P. berghei (18). Immunolocalization of selected DRM-associated proteins indicated that these assemblies may reside in both exported compartments (PVM, MCs) and intracellular membranes/organelles. The analysis of DRM lipids suggested that distinct microdomains exist in the infected erythrocyte that differ in their relative abundance of cholesterol and phospholipids.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones.Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1, 2). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its “effectors” (3), and SNARE³ (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5), termed “cis-SNARE complexes,” if they are all anchored to the same membrane bilayer, or “trans-SNARE complexes” if they are anchored to apposed membranes.Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6), high levels of calcium (7), viral-encoded fusion proteins (8, 9), or SNAREs (10, 11). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12–15), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion.We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17), and key “regulatory” lipids including ERG, phosphoinositides, and DAG (18). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17, 20–22) and proofreads correct vacuolar SNARE pairing (23). HOPS also has direct affinity for phosphoinositides (17). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24). Docking requires Ypt7p (25) and HOPS (17). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26) and lipids (18) required for fusion becomes enriched in a ring-shaped microdomain, the “vertex ring,” which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18, 27). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28), in accord with model liposome studies (14). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies.Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29), this was not stimulated by HOPS and Sec17p/Sec18p (30). SNARE proteoliposomes bearing all the vacuolar lipids (18, 31–33), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS.     

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.HIV-1³ is an enveloped retrovirus, which acquires its lipid envelope by budding from the plasma membrane of the infected host cell. Several reports have shown that the viral membrane is enriched in sphingomyelin (SM), including the unusual sphingolipid dihydrosphingomyelin (DHSM) and collectively referred to as sphingomyelins (SMs), glycosphingolipids, cholesterol (CHOL), saturated phosphoglycerolipids and phosphoinositides (1–4). Moreover the CHOL/phospholipid and protein/lipid ratios of the HIV-1 membrane are high, corresponding to a highly ordered membrane, and are presumed to be different from the overall host cell plasma membrane. Accordingly, the HIV-1 envelope has been considered to be a large raft-like membrane microdomain (3). This is in line with previous reports describing enrichment of raft markers in the HIV-1 membrane and its sensitivity to CHOL-depleting agents (5–9). Furthermore, HIV-1 glycoproteins have been suggested to localize within membrane rafts due to palmitoylation of two cysteines (10), and the main structural Gag protein has been shown to rapidly relocalize to detergent-resistant membranes after initial membrane binding (6).Membrane microdomains are dynamic assemblies resulting from the lateral interaction of lipids and proteins. Two phases coexist in the plasma membrane: the liquid-ordered phase (l_o), mainly composed of CHOL and sphingolipids (SPLs), and the liquid disordered phase (l_d), mainly composed of glycerophospholipids (11–13). In the activated state, l_o microdomains can coalesce and serve as platforms for membrane trafficking, signaling, and virus budding (14, 15). The first method to biochemically enrich membrane rafts was the purification of detergent-resistant membranes, based on their resistance to extraction with non-ionic detergent at 4 °C (16). However, this and other methods based on antibody or cholera toxin binding may lead to artificial aggregation of membrane microdomains and thus do not necessarily represent their native state (17, 18). For these reasons and because the association and dissociation of membrane microdomains appears to occur on a rapid time-scale and the raft size is too small to be optically resolved, the raft concept remains controversial. However, the determination of the HIV-1 lipidome, a native membrane purified without any detergent, has provided strong evidence for the existence of these microdomains (3).Fluorescent lipid analogs that partition preferentially into a specialized lipid phase could be an attractive tool to study membrane microdomains. However, partitioning of such dyes mainly depends on the local chemical environment and not on the phase state of the membrane (19–21). In contrast, Laurdan (6-dodecanoyl-2-dimethylaminonapthalene) is a lipophilic dye that binds to membranes independent of their phase state but reports the phase state by a change in its fluorescence emission (20). Laurdan exhibits a blue shift in its emission spectrum with increasing membrane condensation. This is caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. Thus, excitation of membrane bound Laurdan leads to two emission maxima representing differences in membrane lateral structure. Quantification of membrane order is achieved by computing the Generalized Polarization (GP) value, which is defined as normalized intensity ratio of the two emission channels. GP values range from +1 (most condensed) to −1 (most fluid). They are not biased by probe concentration, membrane ruffles, and surface modifications, such as lipoprotein binding. Furthermore, there is no preferential interaction with a specific lipid, fatty acid, or head group (20, 21). GP value correspondence to different lipid phases was estimated using liposomes with a composition similar to that of cellular membranes (22, 23). Using an equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine, CHOL, and SM as an l_o membrane, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as an l_d and solid ordered (s_o) phase, GP values below +0.25 were shown to correspond to the l_d phase, GP values between +0.25 and +0.5 to the l_o phase, and GP values above +0.5 to the s_o phase (22, 23).Laurdan has been extensively used to characterize domain formation and lateral lipid segregation in model membranes composed of different phospholipid mixtures or lipids extracted from cellular membranes (19, 22–25). It has also been used to study the membrane structure in living cells. Gaus and coworkers observed l_o domains enriched on membrane protrusions (filopodia), adhesion points, and cell-cell contacts (26). They also used Laurdan to address the physical properties of the plasma membrane around the T-cell receptor in activated T cells, observing larger and more stably ordered membrane domains at sites of T-cell activation (27). Quantitative determination of cellular plasma membrane order by fluorescence spectroscopy is complicated due to the rapid internalization and redistribution of the probes to other cellular membranes, making it difficult to interpret the fluorescence measurements over the whole cell. This problem is not encountered in purified virus particles, because they contain only a single membrane. We therefore developed an assay to study viral membrane lateral structure by fluorescence spectroscopy. For this purpose, isogenic HIV-1 particles were produced in two different cell lines, and their GP profiles were determined. In parallel, the lipid constituents were quantified by mass spectrometry. The viral membrane displayed a l_o structure in both cases, but this was more prominent for the virus derived from the T-cell line MT-4 compared with virus derived from 293T cells. The validity of this result was supported by comparing the lipidome of the two viruses, which revealed a significantly higher SMs/phosphatidylcholine (PC) ratio for the MT-4-derived virus. Accordingly, treatment with SM-binding toxins inactivated MT-4-derived virus more efficiently than 293T-derived virus, whereas both viruses exhibited similar infectivities before treatment. The reported approach allows rapid determination of differences in viral membrane order, permitting screening for compounds that perturb l_o domains, which may act as antivirals of a novel type.