Serotonin organelles of rabbit platelets contain synaptophysin

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.     

Intracellular localization of a lipid transfer protein in Vigna unguiculata seeds

Lipid transfer proteins (LTP) facilitate transfer of lipids between membranes in vitro. Up to now, they have been found to be localized basically in the plant cell wall and in compartments linked to lipid metabolism, such as glyoxysomes. Accordingly, LTP are considered to be involved in the plant defence against pathogen microbes and lipid metabolism. We herein show, by immunoelectron microscopy, that besides the cell wall, LTP are localized in the lumen of organelles which we suggest to be the protein storage vacuoles, as well as in vesicles similar to the lipid-containing ones and in the extracellular space of Vigna unguiculata seeds. To further characterize these organelles, we performed subcellular fractionation of membranes isolated from imbibed seeds on a sucrose-density gradient. The analysis of these fractions revealed that the lightest membrane vesicles, derived probably from PSV, contain LTP, α-TIP and K⁺ independent PP_iase, but not γ-TIP and K⁺ stimulated PP_iase. The presence of LTP and vicilins (typical storage protein) in the lumen of these vesicles was confirmed by immunoelectron microscopy. Taken together, the data suggest that the intracellular LTP in the V. unguiculata seeds are localized in protein storage vacuoles and in lipid-containing vesicles.     

Sunday Driver Interacts with Two Distinct Classes of Axonal Organelles

The extreme polarized morphology of neurons poses a challenging problem for intracellular trafficking pathways. The distant synaptic terminals must communicate via axonal transport with the cell soma for neuronal survival, function, and repair. Multiple classes of organelles transported along axons may establish and maintain the polarized morphology of neurons, as well as control signaling and neuronal responses to extracellular cues such as neurotrophic or stress factors. We reported previously that the motor-binding protein Sunday Driver (syd), also known as JIP3 or JSAP1, links vesicular axonal transport to injury signaling. To better understand syd function in axonal transport and in the response of neurons to injury, we developed a purification strategy based on anti-syd antibodies conjugated to magnetic beads to identify syd-associated axonal vesicles. Electron microscopy analyses revealed two classes of syd-associated vesicles of distinct morphology. To identify the molecular anatomy of syd vesicles, we determined their protein composition by mass spectrometry. Gene Ontology analyses of each vesicle protein content revealed their unique identity and indicated that one class of syd vesicles belongs to the endocytic pathway, whereas another may belong to an anterogradely transported vesicle pool. To validate these findings, we examined the transport and localization of components of syd vesicles within axons of mouse sciatic nerve. Together, our results lead us to propose that endocytic syd vesicles function in part to carry injury signals back to the cell body, whereas anterograde syd vesicles may play a role in axonal outgrowth and guidance.     

Lipid rafts,microdomain heterogeneity and inter‐organelle contacts: Impacts on membrane preparation for proteomic studies

In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra‐membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter‐membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.     

A real-time view of life within 100 nm of the plasma membrane

The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.     

High-yield isolation of functionally competent endosomes from mouse lymphocytes.

A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ability to fuse and to acidify in a cell-free system. Endosome fusion was studied by a new assay based on the use of fluorescence resonance energy transfer. This fusion is dependent on ATP and on a cytosolic, thermoresistant but trypsin- and N-ethylmaleimide-sensitive, protein factor. Early endosomes fuse more actively among themselves than with late-endocytic vesicles, and they fuse only slowly with plasma-membrane vesicles.     

The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants.

Soluble proteins are transported to the plant vacuole through the secretory pathway via membrane-bound vesicles. Targeting of vesicles to appropriate organelles requires several membrane-bound and soluble factors that have been characterized in yeast and mammalian systems. For example, the yeast PEP12 protein is a syntaxin homolog that is involved in protein transport to the yeast vacuole. Previously, we isolated an Arabidopsis thaliana homolog of PEP12 by functional complementation of the yeast pep12 mutant. Antibodies raised against the cytoplasmic portion of AtPEP12 have been prepared and used for intracellular localization of this protein. Biochemical analysis indicates that AtPEP12 does not localize to the endoplasmic reticulum, Golgi apparatus, plasma membrane, or tonoplast in Arabidopsis plants; furthermore, based on biochemical and electron microscopy immunogold labeling analyses, AtPEP12 is likely to be localized to a post-Golgi compartment in the vacuolar pathway.     

Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein

The protein 14-3-3 is a key regulator in a cell signaling pathway mediated by protein phosphorylation. To identify the cellular targets of this protein systematically, we have employed a proteomic approach: protein components pulled down from PC12 cells stably expressing a myc-tagged 14-3-3eta isoform were analyzed by means of SDS-PAGE and mass spectrometry. This procedure allowed us to identify more than 30 proteins that include various known and unknown targets of the 14-3-3 protein. Among them are several proteins in the membrane traffic pathway, such as the heavy and light chains (KHC/KIF5B and KLC2) of conventional kinesin, a heterotetrameric mechanochemical motor involved in the ATP-dependent movement of vesicles and organelles along microtubules. Subsequent analysis showed that 14-3-3 directly binds to kinesin heterodimers through interaction with KLC2 and that this interaction is dependent on the phosphorylation of KLC2. Studies on the interaction between 14-3-3 and KLC2 variants expressed in cultured cells coupled with mass spectrometric analysis proved that Ser575 is the site of phosphorylation in KLC2 that is responsible for the in vivo interaction with the 14-3-3 protein. These data add KLC2 to the growing list of 14-3-3 targets, and suggest a role of 14-3-3 in the phosphorylation-regulated cellular transport of vesicles and organelles.     

Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.Abbreviations FITC fluorescein isothiocyanate - IEP Pinner-envelope protein - OE outer-envelope protein - pSSU precursor form of the small subunit of ribulose bisphosphate carboxylaseoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank P. Å. Albertsson, Lund, Sweden, for introducing one of us (S. E.) to the technique of phase partitioning. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246) and Fonds der Chemischen Industrie.     

Proteomic analysis of clathrin-coated vesicles

For more than 50 years cell biologists have embraced the concept that biochemical and enzymatic analysis of isolated subcellular fractions provides insight into the function and machineries of cellular compartments including organelles. The utility of this approach has been significantly enhanced with the advent of mass spectrometry leading to the broad application of organelle proteomics. Clathrin-coated vesicles (CCVs) form at the plasma membrane where they select protein and lipid cargo for endocytic entry into cells. CCVs also form at the trans-Golgi network, where they function in protein transport from the secretory pathway to the endosomal/lysosomal system. Herein we will describe how organelle proteomics of CCVs has greatly expanded our knowledge of the machineries, mechanisms and sites of clathrin-mediated membrane trafficking.     

Evidence that coated vesicles isolated from brain are calcium- sequestering organelles resembling sarcoplasmic reticulum

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Localization of vasopressin in synaptic vesicles of extra-hypothalamic rat brain

The subcellular localization of vasopressin (VP) from extra-hypothalamic areas of rat brain was investigated by measuring its distribution (a) along a continuous sucrose gradient; (b) during the preparation of isolated nerve endings (synaptosomes) and (c) during the preparation of synaptic vesicles.Quite large amounts of vasopressin are isolated in the same fractions as mitochondria, as well as synaptosomes. Osmotic rupture of membrane bound organelles in the homogenate results in the vasopressin being measured largely in the fraction containing synaptic vesicles. These results would suggest that vasopressin could be released by nerve terminals which is consistent with the hypothesis that it may have a neurotransmitter/neuromodulator function in the CNS.     

Lipid-rich tapetosomes in Brassica tapetum are composed of oleosin-coated oil droplets and vesicles, both assembled in and then detached from the endoplasmic reticulum

Tapetosomes are abundant organelles in tapetum cells of floral anthers in Brassicaceae species. They contain triacylglycerols (TAGs), the amphipathic protein oleosins and putative vesicles and play a predominant role in pollen-coat formation. Here we report the biogenesis and structures of tapetosomes in Brassica. Immunofluorescence confocal microscopy revealed that during early anther development, the endoplasmic reticulum (ER) luminal protein calreticulin existed as a network in tapetum cells, which contained no oleosins. Subsequently, oleosins appeared together with calreticulin in the ER network, which possessed centers with a higher ratio of oleosin to calreticulin. Finally, the ER network largely disappeared, and solitary tapetosomes containing oleosins and calreticulin became abundant. Transmission electron microscopy also revealed a close association between a maturing tapetosome and numerous ER cisternae. Mature, solitary tapetosomes were isolated and found to contain oleosins, calreticulin and the ER luminal binding protein (BiP). Isolated tapetosomes were treated with sodium carbonate and subfractionated by centrifugation. Two morphologically distinct constituents were isolated: low-density oil droplets, which contained oleosins and TAGs, and relatively high-density cisternae-like vesicles, which possessed calreticulin and BiP. Thus, tapetosomes are composed of oleosin-coated oil droplets and vesicles, both of which are assembled in and then detached from the ER. The structure and biogenesis of tapetosomes are unique among eukaryotic organelles. After tapetum cells lyzed, oleosins but not calreticulin and BiP of tapetosomes were transferred to the pollen surface.     

Plant coated vesicles

Abstract. Coated vesicles are organelles frequently encountered in many plant cell types often in association with the plasma membrane, Golgi apparatus, partially coated reticulum and multivesicular bodies. They are readily identified by a characteristic cage or basket composed of interlocking triskelions of the protein clathrin which are bound to the surface of the vesicle membrane. Although their transport function has been well studied and characterized in mammalian systems, the possible importance of coated vesicles as transport organelles in plant cells is only just beginning to be explored. In this review, the authors describe the structure of higher plant coated vesicles and discuss their possible involvement in the endocytosis of marcromolecules, in exocytosis and in the intracellular transport of material between cytoplasmic compartments. Their possible role in maintaining the macromolecular composition of the plasma membrane whilst allowing recycling of excess lipid bilayer and their potential application as vehicles for the introduction of foreign macromolecules into plant cells are discussed.     

Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.     

Interactions of pathogen‐containing compartments with the secretory pathway

A subgroup of intracellular pathogens reside and replicate within membrane‐bound compartments often termed pathogen‐containing compartments (PCC). PCCs navigate around a wide range of host cell vesicles and organelles. In light of the perils of engaging with vesicles of the endocytic pathway, most PCCs modulate their interactions with endocytic vesicles while a few avoid those interactions. The secretory pathway constitutes another important grouping of vesicles and organelles in host cells. Although the negative consequences of engaging with the secretory pathway are not known, there is evidence that PCCs interact differentially with vesicles and organelles in this pathway as well. In this review, we consider three prokaryote pathogens and two protozoan parasites for which there is information on the interactions of their PCCs with the secretory pathway. Current understandings of the molecular interactions as well as the metabolic benefits that accompany those interactions are discussed. Not unexpectedly, our understanding of the extent of these interactions is variable. An underlying theme that is brought to the fore is that PCCs establish preferential interactions with distinct compartments of the secretory pathway.     

Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.     

Cholinergic synaptic vesicles from Torpedo marmorata contain an atractyloside-binding protein related to the mitochondrial ADP/ATP carrier

Atractyloside is known to bind to the ADP/ATP translocase of the inner mitochondrial membrane, a complex formed by two basic protein subunits of relative molecular mass around 30 000. We found that synaptic vesicles from the electric organ of Torpedo marmorata, which store acetylcholine and ATP, bind atractyloside as well. Similarly to mitochondria, a protein-atractyloside complex could be solubilized from vesicle membranes with Triton X-100. Characterization of the complex by gel filtration, isoelectric focusing and gel electrophoresis revealed that atractyloside was bound to protein V11, earlier described as a major vesicle membrane component with a relative molecular mass around 34 000 and a basic isoelectric point. Since earlier experiments have already shown that uptake of ATP into isolated vesicles in vitro is inhibited by atractyloside, we can conclude now that V11 constitutes the nucleotide carrier of this secretory organelle. The structural and functional relationship of the mitochondrial and vesicular nucleotide translocases suggest a common evolutionary origin.