Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein

The function of the guanosine triphosphate (GTP)-binding protein Ypt1 in regulating vesicular traffic was studied in a cell-free system that reconstitutes transport from the endoplasmic reticulum to the Golgi. Blocking the Ypt1 protein activity resulted in accumulation of vesicles that act as an intermediate passing between the two compartments. The Ypt1 protein was found on the outer side of these vesicles. The transport process is completed by fusion of these vesicles with the acceptor compartment, and Ypt1 protein activity was needed for this step. Thus, a specific GTP-binding protein is required for either attachment or fusion (or both) of secretory vesicles with the acceptor compartment during protein secretion.     

Control of local protein synthesis and initial events in myelination by action potentials

Formation of myelin, the electrical insulation on axons produced by oligodendrocytes, is controlled by complex cell-cell signaling that regulates oligodendrocyte development and myelin formation on appropriate axons. If electrical activity could stimulate myelin induction, then neurodevelopment and the speed of information transmission through circuits could be modified by neural activity. We find that release of glutamate from synaptic vesicles along axons of mouse dorsal root ganglion neurons in culture promotes myelin induction by stimulating formation of cholesterol-rich signaling domains between oligodendrocytes and axons, and increasing local synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn kinase-dependent signaling. This axon-oligodendrocyte signaling would promote myelination of electrically active axons to regulate neural development and function according to environmental experience.     

Asymmetric tethering of flat and curved lipid membranes by a golgin

Golgins, long stringlike proteins, tether cisternae and transport vesicles at the Golgi apparatus. We examined the attachment of golgin GMAP-210 to lipid membranes. GMAP-210 connected highly curved liposomes to flatter ones. This asymmetric tethering relied on motifs that sensed membrane curvature both in the N terminus of GMAP-210 and in ArfGAP1, which controlled the interaction of the C terminus of GMAP-210 with the small guanine nucleotide-binding protein Arf1. Because membrane curvature constantly changes during vesicular trafficking, this mode of tethering suggests a way to maintain the Golgi architecture without compromising membrane flow.     

Transport studies in bacterial membrane vesicles

The use of bacterial membrane vesicles as an experimental system for the study of active transport has been discussed. Vesicles are prepared from osmotically sensitized bacteria, and consist of osmotically intact, membranebound sacs without internal structure. They retain litle or no cytoplasm. Under appropriate conditions, these vesicles catalyze the transport of a variety of solutes at rates which are comparable, in many cases, to those of intact cells. Two general types of transport systems have been elucidated in the vesicle system: (i) group translocation systems which catalyze vectorial covalent reactions; and (ii) respirationlinked transport systems that catalyze the active transport of a whole range of metabolites against an electrochemical or osmotic gradient. In E. coli membrane vesicles, the respiration-linked transport systems are coupled primarily to the oxidation of (D)-lactate to pyruvate, catalyzed by a flavin-linked, membrane-bound (D)-lactate dehydrogenase which has been purified to homogeneity. Electrons derived from (D)-lactate or certain artificial electron donors are transferred to oxygen by means of a membrane-bound respiratory chain, and respiration is coupled to active transport within a segment of the respiratory chain between the primary dehydrogenase and cytochrome. b(l). The great majority of the individual membrane vesicles in the population catalyze active transport, and the generation or hydtolysis of ATP is not involved. Under anaerobic conditions, fumarate or nitrate can be utilized in place of oxygen as terminal electron acceptors. With the exception that (D)-lactate is not always the most effective electron donor for active transport, vesicles prepared from a number of other organisms catalyze transport in a similar manner. Fluorescent dansylgalactosides are useful molecular probes of active transport in the vesicle system. These compounds are competitive inhibitors of beta-galactoside transport, but are not transported themselves. Fluorescence studies indicate that the lac carrier protein constitutes approximately 3 to 6 percent of the total membrane protein, and that it is not accessible to the external medium unless the membrane is "energized." Thus, energy is coupled to one of the initial steps in the transport process. Studies with a photoaffinity-labeled galactoside provide independent support for this conclusion. When membrane vesicles prepared from a (D)-lactate dehydrogenase mutant of E. coli are treated with (D)-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze (D)-lactate oxidation and (D)-lactate-dependent active transport. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wildtype vesicles. Titration studies with dansylgalactoside demonstrate that there is at least a seven- to eightfold excess of lac carrier protein relative to (D)-lactate dehydrogenase. Evidence is presented indicating that the enzyme is bound to the inner surface of native membrane vesicles and to the outer surface of reconstituted vesicles, and that the flavin coenzyme moiety is critically involved in binding. Possible mechanisms of respirationlinked active transport are discussed.     

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset of excitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.     

Astrocytes potentiate transmitter release at single hippocampal synapses

Astrocytes play active roles in brain physiology. They respond to neurotransmitters and modulate neuronal excitability and synaptic function. However, the influence of astrocytes on synaptic transmission and plasticity at the single synapse level is unknown. Ca(2+) elevation in astrocytes transiently increased the probability of transmitter release at hippocampal area CA3-CA1 synapses, without affecting the amplitude of synaptic events. This form of short-term plasticity was due to the release of glutamate from astrocytes, a process that depended on Ca(2+) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein and that activated metabotropic glutamate receptors (mGluRs). The transient potentiation of transmitter release became persistent when the astrocytic signal was temporally coincident with postsynaptic depolarization. This persistent plasticity was mGluR-mediated but N-methyl-d-aspartate receptor-independent. These results indicate that astrocytes are actively involved in the transfer and storage of synaptic information.     

Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.     

Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport

Experiments with vesicles containing N-methyl-D-aspartate (NMDA) receptor 2B (NR2B subunit) show that they are transported along microtubules by KIF17, a neuron-specific molecular motor in neuronal dendrites. Selective transport is accomplished by direct interaction of the KIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is a constituent of a large protein complex including mLin-2 (CASK), mLin-7 (MALS/Velis), and the NR2B subunit. This interaction, specific for a neurotransmitter receptor critically important for plasticity in the postsynaptic terminal, may be a regulatory point for synaptic plasticity and neuronal morphogenesis.     

Methylation increases sodium transport into A6 apical membrane vesicles: possible mode of aldosterone action

When isolated apical membrane vesicles prepared from cultured A6 epithelia were incubated in vitro with the methyl donor S-adenosylmethionine, the control rate of amiloride-inhibitable sodium transport was doubled. The methylation inhibitors 3-deazaadenosine and S-adenosyl homocysteine returned the S-adenosyl-methionine-stimulated sodium transport to control levels. Neither these agents nor adenosine affected sodium transport into control vesicles. In vesicles incubated with S-adenosyl-[3H-methyl]methionine, both membrane phospholipids and proteins were labeled, and this labeling was inhibited by deazaadenosine. In vesicles prepared from A6 cells treated with aldosterone, sodium transport was twice the control value and S-adenosylmethionine did not cause any further stimulation of transport. In those vesicles, both lipid and protein methylation were increased. These results suggest that methylation, which increases the rate of amiloride-sensitive sodium transport is involved in the action of aldosterone at the apical membrane level in epithelia.     

SNARE function analyzed in synaptobrevin/VAMP knockout mice

SNAREs (soluble NSF-attachment protein receptors) are generally acknowledged as central components of membrane fusion reactions, but their precise function has remained enigmatic. Competing hypotheses suggest roles for SNAREs in mediating the specificity of fusion, catalyzing fusion, or actually executing fusion. We generated knockout mice lacking synaptobrevin/VAMP 2, the vesicular SNARE protein responsible for synaptic vesicle fusion in forebrain synapses, to make use of the exquisite temporal resolution of electrophysiology in measuring fusion. In the absence of synaptobrevin 2, spontaneous synaptic vesicle fusion and fusion induced by hypertonic sucrose were decreased approximately 10-fold, but fast Ca2+-triggered fusion was decreased more than 100-fold. Thus, synaptobrevin 2 may function in catalyzing fusion reactions and stabilizing fusion intermediates but is not absolutely required for synaptic fusion.     

The biochemistry of memory: a new and specific hypothesis

Recent studies have uncovered a synaptic process with properties required for an intermediate step in memory storage. Calcium rapidly and irreversibly increases the number of receptors for glutamate (a probable neurotransmitter) in forebrain synaptic membranes by activating a proteinase (calpain) that degrades fodrin, a spectrin-like protein. This process provides a means through which physiological activity could produce long-lasting changes in synaptic chemistry and ultrastructure. Since the process is only poorly represented in the brain stem, it is hypothesized to be responsible for those forms of memory localized in the telencephalon.     

Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane

The quaternary structure and functional properties of synaptophysin, a major integral membrane protein of small presynaptic vesicles, were investigated. Cross-linking and sedimentation studies indicate that synaptophysin is a hexameric homo-oligomer, which in electron micrographs exhibits structural features common to channel-forming proteins. On reconstitution into planar lipid bilayers, purified synaptophysin displays voltage-sensitive channel activity with an average conductance of about 150 picosiemens. Because specific channels and fusion pores have been implicated in vesicular uptake and release of secretory compounds, synaptophysin may have a role in these processes.     

Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression

A hippocampal pyramidal neuron receives more than 10(4) excitatory glutamatergic synapses. Many of these synapses contain the molecular machinery for messenger RNA translation, suggesting that the protein complement (and thus function) of each synapse can be regulated on the basis of activity. Here, local postsynaptic protein synthesis, triggered by synaptic activation of metabotropic glutamate receptors, was found to modify synaptic transmission within minutes.     

A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions

Complementary DNA and genomic clones were isolated and sequenced corresponding to rat and human synaptophysin (p38), a major integral membrane protein of synaptic vesicles. The deduced amino acid sequences indicate an evolutionarily highly conserved protein that spans the membrane four times. Both amino and carboxyl termini face the cytoplasm, with the latter containing ten copies of a tyrosine-rich pentapeptide repeat. The structure of synaptophysin suggests that the protein may function as a channel in the synaptic vesicle membrane, with the carboxyl terminus serving as a binding site for cellular factors.     

Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.     

A test of clathrin function in protein secretion and cell growth

Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible. The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion. Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11. Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA. Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains. Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate. Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles.     

Morphologic alterations of synapses in electrically stimulated superior cervical ganglia of the cat

Prolonged preganglionic stimulation produces marked ultrastructural changes in presynaptic endings, which develop larger zones of contact with post-synaptic dendrites. Profiles of such endings, compared to controls, have fewer synaptic vesicles, similar areas, and greater circumferences. These results are compatible with the hypothesis that synaptic vesicles become incorporated into the plasma membrane during stimulation.     

The role of GTP-binding protein activity in fast central synaptic transmission

Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are involved in exocytosis, endocytosis, and recycling of vesicles in yeast and mammalian secretory cells. However, little is known about their contribution to fast synaptic transmission. We loaded guanine nucleotide analogs directly into a giant nerve terminal in rat brainstem slices. Inhibition of G-protein activity had no effect on basal synaptic transmission, but augmented synaptic depression and significantly slowed recovery from depression. A nonhydrolyzable GTP analog blocked recovery of transmission from activity-dependent depression. Neither effect was accompanied by a change in presynaptic calcium currents. Thus, G proteins contribute to fast synaptic transmission by refilling synaptic vesicles depleted after massive exocytosis.     

Postsynaptic signaling and plasticity mechanisms

In excitatory synapses of the brain, specific receptors in the postsynaptic membrane lie ready to respond to the release of the neurotransmitter glutamate from the presynaptic terminal. Upon stimulation, these glutamate receptors activate multiple biochemical pathways that transduce signals into the postsynaptic neuron. Different kinds of synaptic activity elicit different patterns of postsynaptic signals that lead to short- or long-lasting strengthening or weakening of synaptic transmission. The complex molecular mechanisms that underlie postsynaptic signaling and plasticity are beginning to emerge.     

Long-term synaptic potentiation

Long-term synaptic potentiation (LTP) is a leading candidate for a synaptic mechanism of rapid learning in mammals. LTP is a persistent increase in synaptic efficacy that can be quickly induced. The biophysical process that controls one type of LTP is formally similar to a synaptic memory mechanism postulated decades ago by the psychologist Donald Hebb. A key aspect of the modification process involves the N-methyl-D-aspartate (NMDA) receptor-ionophore complex. This ionophore allows calcium influx only if the endogenous ligand glutamate binds to the NMDA receptor and if the voltage across the associated channel is also sufficiently depolarized to relieve a magnesium block. According to one popular hypothesis, the resulting increase in the intracellular calcium concentration activates protein kinases that enhance the postsynaptic conductance. Further biophysical and molecular understanding of the modification process should facilitate detailed explorations of the mnemonic functions of LTP.