scholarly journals Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

1997 ◽  
Vol 139 (4) ◽  
pp. 917-927 ◽  
Author(s):  
Anne E. West ◽  
Rachael L. Neve ◽  
Kathleen M. Buckley
Keyword(s):  
Amino Acid ◽  
Synaptic Vesicle ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Amino Acid Region ◽  
Human Transferrin Receptor ◽  
Cultured Hippocampal Neurons

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.

scholarly journals Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

1990 ◽  
Vol 110 (2) ◽  
pp. 283-294 ◽  
Author(s):  
S Q Jing ◽  
T Spencer ◽  
K Miller ◽  
C Hopkins ◽  
I S Trowbridge
Keyword(s):  
Amino Acid ◽  
Transferrin Receptor ◽  
High Efficiency ◽  
Signal Sequence ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Human Transferrin ◽  
Amino Acid Region ◽  
Human Transferrin Receptor ◽  
Mutant Receptors

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

scholarly journals BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin–β-catenin interactions

2006 ◽  
Vol 174 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Shernaz X. Bamji ◽  
Beatriz Rico ◽  
Nikole Kimes ◽  
Louis F. Reichardt
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Time Lapse ◽  
Synapse Density ◽  
Vesicle Mobility ◽  
Small Clusters ◽  
Vertebrate Central Nervous System

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin–β-catenin adhesion complexes that occurs after tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin–β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin–β-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

scholarly journals Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain.

1990 ◽  
Vol 1 (4) ◽  
pp. 369-377 ◽  
Author(s):  
T E McGraw ◽  
F R Maxfield
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rate Constant ◽  
Transferrin Receptor ◽  
Aromatic Amino Acid ◽  
Opposite Polarity ◽  
Receptor Internalization ◽  
Aromatic Side Chain ◽  
Human Transferrin ◽  
Human Transferrin Receptor

The objective of this work is to identify the elements of the human transferrin receptor that are involved in receptor internalization, intracellular sorting, and recycling. We have found that an aromatic side chain at position 20 on the cytoplasmic portion of the human transferrin receptor is required for efficient internalization. The wild-type human transferrin receptor has a tyrosine at this position. Replacement of the Tyr-20 with an aromatic amino acid does not alter the rate constant of internalization, whereas substitution with the nonaromatic amino acids serine, leucine, or cysteine reduces the internalization rate constant approximately three-fold. These results are consistent with similar studies of other receptor systems that have also documented the requirement for a tyrosine in rapid internalization. The amino terminus of the transferrin receptor is cytoplasmic, with the tyrosine 41 amino acids from the membrane. These two features distinguish the transferrin receptor from the other membrane proteins for which the role of tyrosine in internalization has been examined, because these proteins have the opposite polarity with respect to the membrane and because the tyrosines are located closer to the membrane (within 25 amino acids). The externalization rate for the recycling of the transferrin receptor is not altered by any of these substitutions, demonstrating that the aromatic amino acid internalization signal is not required for the efficient exocytosis of internalized receptor.

Expression of amino acid sequences of the chromogranin A molecule and synaptic vesicle protein 2 in neuroendocrine tumors of the lung

2005 ◽  
Vol 446 (6) ◽  
pp. 604-612 ◽  
Author(s):  
Guida Maria Portela-Gomes ◽  
Lars Grimelius ◽  
Mats Stridsberg ◽  
Enrica Bresaola ◽  
Giuseppe Viale ◽  
...  
Keyword(s):  
Amino Acid ◽  
Synaptic Vesicle ◽  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Amino Acid Sequences ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein

scholarly journals Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 580-586 ◽  
Author(s):  
EA Rutledge ◽  
BJ Root ◽  
JJ Lucas ◽  
CA Enns
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Transferrin Receptor ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Soluble Form ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
Human Transferrin Receptor ◽  
Intracellular Compartments

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

A comparative study of repeated sequences in the SM50 gene of some sea urchins

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S75-S75
Author(s):  
Masayuki Goto ◽  
Masahiro Matsumoto ◽  
Takashi Kitajima ◽  
Akiya Hino
Keyword(s):  
Amino Acid ◽  
Sea Urchin ◽  
Sequence Data ◽  
Sea Urchins ◽  
Rabbit Antiserum ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Immunofluorescent Staining ◽  
Upstream Region ◽  
Amino Acid Region

Spicule matrix proteins of sea urchin embryo are the specific products of the micromere / primary mesenchyme cell (PMC) lineage, and are considered to be involved in spicule formation (Wilt, 1999). One of these proteins, SM50, has been described for three species: Strongylocentrotus purpuratus (SP), Lytechinus pictus (Lp) and Hemicentrotus pulcherrimus (Hp) (for references see Wilt, 1999). The nucleotide and amino acid sequences are well conserved in these species. SM50 proteins of these species have repetitive amino acid sequences in the carboxyl-terminal half of the proteins. Therefore, examination of SM50 sequences, especially the repetitive sequence region, in various species will help an understanding of the process of sea urchin ontogeny and evolution. In this study we tried to amplify, by PCR, the SM50 sequences of species for which no sequence data are reported.Total DNA was extracted from the sperm of sea urchins by standard procedures. The purified DNA was subjected to PCR to amplify the repetitive amino acid region and its upstream region. The primers were designed based on the highly conserved sequences in the reported SM50 as Consensus-Degenerate Hybrid Oligonucleotide Primers (Rose et al., 1997). The amplified products were gel-purified, and sequenced using ABI PRISM 310 Genetic Analyzer using PCR primers. The determined nucleotide sequences were translated into amino acid sequences and compared among species with a phylogenetic tree constructed by the neighbour-joining method. For indirect immunofluorescent staining, embryos were fixed with 70% methanol and reacted with rabbit antiserum against recombinant SM50 protein.

scholarly journals Conservation of the Conformation and Positive Charges of Hepatitis C Virus E2 Envelope Glycoprotein Hypervariable Region 1 Points to a Role in Cell Attachment

2001 ◽  
Vol 75 (12) ◽  
pp. 5703-5710 ◽  
Author(s):  
François Penin ◽  
Christophe Combet ◽  
Georgios Germanidis ◽  
Pierre-Olivier Frainais ◽  
Gilbert Deléage ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Entry ◽  
Envelope Glycoprotein ◽  
Cell Attachment ◽  
Hypervariable Region ◽  
Amino Acid Sequences ◽  
Specific Sequence ◽  
Amino Acid Region

ABSTRACT Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.

scholarly journals Synaptophysin I Controls the Targeting of VAMP2/Synaptobrevin II to Synaptic Vesicles

2003 ◽  
Vol 14 (12) ◽  
pp. 4909-4919 ◽  
Author(s):  
Maria Pennuto ◽  
Dario Bonanomi ◽  
Fabio Benfenati ◽  
Flavia Valtorta
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Cell Body ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Direct Interaction ◽  
Diffuse Component ◽  
Synaptotagmin I ◽  
Dose Dependent

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.

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