The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain-binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Bordetella bronchiseptica genetics growth & development metabolism MeSH
- Cell Membrane metabolism MeSH
- Phagocytosis MeSH
- Phospholipids metabolism MeSH
- HeLa Cells MeSH
- Humans MeSH
- Lipid Bilayers metabolism MeSH
- Membrane Microdomains metabolism MeSH
- Protein Domains MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.
- MeSH
- Arabidopsis cytology metabolism MeSH
- Brassinosteroids pharmacology MeSH
- Spatio-Temporal Analysis * MeSH
- Diffusion MeSH
- Endocytosis drug effects MeSH
- Clathrin metabolism MeSH
- Membrane Microdomains drug effects metabolism MeSH
- Protein Multimerization drug effects MeSH
- Motion MeSH
- Protein Kinases metabolism MeSH
- Arabidopsis Proteins metabolism MeSH
- Plant Cells drug effects metabolism MeSH
- Signal Transduction drug effects MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.
- MeSH
- Enzyme Activation MeSH
- Lymphocyte Activation MeSH
- Leukocyte Common Antigens metabolism MeSH
- Cell Membrane metabolism MeSH
- CD4-Positive T-Lymphocytes immunology MeSH
- Centrifugation, Density Gradient MeSH
- Detergents pharmacology MeSH
- Membrane Microdomains metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Signal Transduction MeSH
- Protein Transport MeSH
- Lymphocyte Specific Protein Tyrosine Kinase p56(lck) immunology metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.
- MeSH
- Bacterial Proteins metabolism MeSH
- Photosynthesis physiology MeSH
- Photosystem I Protein Complex metabolism MeSH
- Photosystem II Protein Complex metabolism MeSH
- Phycobilisomes metabolism MeSH
- Microscopy, Confocal MeSH
- Membrane Microdomains metabolism MeSH
- Synechocystis MeSH
- Thylakoids metabolism MeSH
- Imaging, Three-Dimensional MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Transmembrane adaptor proteins (TRAPs) are structurally related proteins that have no enzymatic function, but enable inducible recruitment of effector molecules to the plasma membrane, usually in a phosphorylation dependent manner. Numerous surface receptors employ TRAPs for either propagation or negative regulation of the signal transduction. Several TRAPs (LAT, NTAL, PAG, LIME, PRR7, SCIMP, LST1/A, and putatively GAPT) are known to be palmitoylated that could facilitate their localization in lipid rafts or tetraspanin enriched microdomains. This review summarizes expression patterns, binding partners, signaling pathways, and biological functions of particular palmitoylated TRAPs with an emphasis on the three most recently discovered members, PRR7, SCIMP, and LST1/A. Moreover, we discuss in silico methodology used for discovery of new family members, nature of their binding partners, and microdomain localization.
- MeSH
- Adaptor Proteins, Signal Transducing metabolism MeSH
- Leukocytes metabolism physiology MeSH
- Humans MeSH
- Lipoylation MeSH
- Membrane Microdomains metabolism MeSH
- Membrane Proteins metabolism MeSH
- Signal Transduction * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.
- MeSH
- Arabidopsis genetics metabolism MeSH
- Cysteine genetics MeSH
- Conserved Sequence * MeSH
- Plant Roots growth & development metabolism MeSH
- Indoleacetic Acids metabolism MeSH
- Membrane Microdomains metabolism MeSH
- Arabidopsis Proteins chemistry genetics metabolism MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
Adenylate cyclase toxin (CyaA) of Bordetella pertussis penetrates the membrane of eukaryotic cells, producing high levels of intracellular cAMP, as well as hemolysis that results from the formation of cation-selective toxin channels in the membrane. Using several microscopical approaches we studied the effects of CyaA action on the morphology of sheep erythrocytes during early phases preceding lysis and examined localization of CyaA molecules within the erythrocyte membrane. CyaA induced a cascade of morphological changes of erythrocytes, such as shrinkage, formation of membrane projections, and blebs and swelling. The use of an enzymatically inactive CyaA-AC- toxoid that is unable to produce cAMP and of a CyaA-E581K mutant exhibiting higher hemolytic activity than with CyaA showed that the hemolytic activity is responsible for the induction of morphological changes of erythrocytes. Further, immunolabeling of inserted CyaA-232/FLAG molecules with specific anti-FLAG antibodies and IgG-gold particles indicated a clustered distribution of CyaA molecules in erythrocyte membrane. This was confirmed by immunofluorescence and confocal microscopy, which revealed uniform stoichiometry of CyaA clusters, suggesting CyaA binding into specific domains in erythrocyte membrane. Indeed, a decrease of CyaA binding after cholesterol depletion of erythrocytes suggests toxin targeting and binding to membrane microdomains (rafts). Copyright 2006 Wiley-Liss, Inc.
- MeSH
- Adenylate Cyclase Toxin analysis toxicity MeSH
- Erythrocyte Membrane chemistry ultrastructure MeSH
- Erythrocytes chemistry drug effects ultrastructure MeSH
- Financing, Organized MeSH
- Microscopy, Fluorescence MeSH
- Hemolysis MeSH
- Microscopy, Immunoelectron MeSH
- Immunohistochemistry MeSH
- Microscopy, Confocal MeSH
- Membrane Microdomains chemistry metabolism MeSH
- Microscopy, Electron, Scanning Transmission MeSH
- Mutation MeSH
- Sheep MeSH
- Amino Acid Substitution MeSH
- Toxoids metabolism MeSH
- Microscopy, Electron, Transmission MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6 is an extensively posttranslationally modified transmembrane protein and that N- and O-glycosylations of amino acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS polyacrylamide gels. Site-directed mutagenesis confirmed that all six extracellular asparagines are N-glycosylated and that the Ser/Thr/Pro cluster in the "stalk" domain juxtaposed to the cysteine-rich domains (CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368 is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.
- MeSH
- Cell Line MeSH
- Glycosylation MeSH
- HeLa Cells MeSH
- HL-60 Cells MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Lipoylation MeSH
- Membrane Microdomains metabolism MeSH
- Molecular Weight MeSH
- Mutagenesis, Site-Directed MeSH
- Cell Line, Tumor MeSH
- Protein Processing, Post-Translational MeSH
- Protein Isoforms physiology genetics chemistry MeSH
- Receptors, Tumor Necrosis Factor genetics chemistry metabolism MeSH
- Recombinant Proteins genetics chemistry metabolism MeSH
- Sequence Deletion MeSH
- Protein Structure, Tertiary MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome's proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.
The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.
- MeSH
- Enzyme Activation drug effects genetics immunology MeSH
- Antigens immunology metabolism pharmacology MeSH
- Phosphorylation drug effects genetics immunology MeSH
- Enzyme Inhibitors pharmacology MeSH
- Rats MeSH
- Mast Cells immunology metabolism MeSH
- Membrane Microdomains genetics immunology metabolism MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Oxidation-Reduction drug effects MeSH
- Oxidants pharmacology MeSH
- Hydrogen Peroxide pharmacology MeSH
- Receptors, IgE genetics immunology metabolism MeSH
- src-Family Kinases genetics immunology metabolism MeSH
- Protein Transport drug effects genetics immunology MeSH
- Protein Tyrosine Phosphatases antagonists & inhibitors genetics immunology metabolism MeSH
- Vanadates pharmacology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
