Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

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When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted . This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation . At 1 min of stimulation, the 30 0/0 depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis . After 15 min of stimulation, the 607, depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae . When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles . When muscles were soaked in horseradish peroxidase (HRP), this tracer first entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles . When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring . Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae . In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface . Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae .

Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.

At 4°C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37°C receptor-mediated endocytosis of transferrin occurred. Uptake at 37°C exceeded binding at 4°C by 2.5-fold and saturated after 20-30 min. During uptake at 37°C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4°C destroyed surface receptors, but with subsequent incubation at 37°C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37°C colloidal goldtransferrin (Auto clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37°C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatasenegative compartments. AuTf, like transferrin, was released with subsequent incubation at 37°C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.Receptor-mediated binding and endocytosis of transferrin occur in many cell types (4,17,23,24,34,38) and appear to be requisite steps in iron delivery under some conditions (7, 9). Transferrin uptake has been best studied in erythropoietic cells, where the synthesis of hemoglobin requires a large amount of iron. Expression of transferrin receptors in these cells peaks early in development and declines progressively during the maturation of erythroblasts and reticulocytes (23,34,36). Despite their lower level of receptor expression, reticulocytes are a convenient model system, since they may be easily isolated from the blood of anemic animals.Transferrin binding is mediated by protease-sensitive receptors (8) but is not inhibited by glycosidase treatment of either transferrin or its receptor (8,18,22). Transferrin receptors have been identified and characterized by many research groups. The uptake of transferrin is both temperature and energy dependent, and transferrin endocytosis has been demonstrated by the use of EM-autoradiography (9, 21), ferritinor horseradish peroxidase-conjugated transferrin (9, 31), and ferritin-conjugated antitransferrin antibodies (31 ). These techniques reveal that transferrin binds to the plasma membrane at 4"C and appears inside intracellular vesicles during incubation at 37"C. Receptor-mediated transferrin uptake may occur by mechanisms ...