Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells.
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Synaptogyrins constitute a family of synaptic vesicle proteins of unknown function. With the full-length structure of a new brain synaptogyrin isoform, we now show that the synaptogyrin family in vertebrates includes two neuronal and one ubiquitous isoform. All of these synaptogyrins are composed of a short conserved N-terminal cytoplasmic sequence, four homologous transmembrane regions, and a variable cytoplasmic C-terminal tail that is tyrosine-phosphorylated. The localization, abundance, and conservation of synaptogyrins suggest a function in exocytosis. To test this, we employed a secretion assay in PC12 cells expressing transfected human growth hormone (hGH) as a reporter protein. When Ca2+-dependent hGH secretion from PC12 cells was triggered by high K+ or alpha-latrotoxin, co-transfection of all synaptogyrins with hGH inhibited hGH exocytosis as strongly as co-transfection of tetanus toxin light chain. Synaptophysin I, which is distantly related to synaptogyrins, was also inhibitory but less active. Inhibition was independent of the amount of hGH expressed but correlated with the amount of synaptogyrin transfected. Inhibition of exocytosis was not observed with several other synaptic proteins, suggesting specificity. Analysis of the regions of synaptogyrin required for inhibition revealed that the conserved N-terminal domain of synaptogyrin is essential for inhibition, whereas the long C-terminal cytoplasmic tail is largely dispensable. Our results suggest that synaptogyrins are conserved components of the exocytotic apparatus, which function as regulators of Ca2+-dependent exocytosis. (+info)
Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I.
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We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself. (+info)
Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles.
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Although Glut4 traffic is routinely described as translocation from an "intracellular storage pool" to the plasma membrane, it has been long realized that Glut4 travels through at least two functionally distinct intracellular membrane compartments on the way to and from the cell surface. Biochemical separation and systematic studies of the individual Glut4-containing compartments have been limited by the lack of appropriate reagents. We have prepared a monoclonal antibody against a novel component protein of Glut4 vesicles and have identified this protein as cellugyrin, a ubiquitously expressed homologue of a major synaptic vesicle protein, synaptogyrin. By means of sucrose gradient centrifugation, immunoadsorption, and confocal microscopy, we have shown that virtually all cellugyrin is co-localized with Glut4 in the same vesicles. However, unlike Glut4, cellugyrin is not re-distributed to the plasma membrane in response to insulin stimulation, and at least 40-50% of the total population of Glut4 vesicles do not contain this protein. We suggest that cellugyrin represents a specific marker of a functionally distinct population of Glut4 vesicles that permanently maintains its intracellular localization and is not recruited to the plasma membrane by insulin. (+info)
A conserved mechanism of synaptogyrin localization.
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We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-tagged Caenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates. (+info)
Isolation and characterization of the two major intracellular Glut4 storage compartments.
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In rat adipose cells, intracellular Glut4 resides in two distinct vesicular populations one of which contains cellugyrin whereas another lacks this protein (Kupriyanova, T. A., and Kandror, K. V. (2000) J. Biol. Chem. 275, 36263--36268). Cell surface biotinylated MPR and (125)I-labeled transferrin are accumulated in cellugyrin-positive vesicles and to a lesser extent in cellugyrin-negative vesicles. An average cellugyrin-positive vesicle carries not more than one molecule of either Glut4, insulin-responsive aminopeptidase (IRAP), or transferrin receptor (TfR), whereas cellugyrin-negative vesicles contain five to six molecules of Glut4, more than 10 molecules of IRAP, and still one molecule of TfR per vesicle. Cellugyrin-negative vesicles are translocated to the cell surface after insulin stimulation, whereas cellugyrin-positive vesicles maintain intracellular localization both in the absence and in the presence of insulin and, therefore, may be involved in interendosomal protein transport. Both cellugyrin-positive and cellugyrin-negative vesicles are present in extracts of non-homogenized cells and therefore may represent the major form of Glut4 storage in vivo. (+info)
Cellugyrin and synaptogyrin facilitate targeting of synaptophysin to a ubiquitous synaptic vesicle-sized compartment in PC12 cells.
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Cellugyrin represents a ubiquitously expressed four-transmembrane domain protein that is closely related to synaptic vesicle protein synaptogyrin and, more remotely, to synaptophysin. We report here that, in PC12 cells, cellugyrin is localized in synaptic-like microvesicles (SLMVs), along with synaptogyrin and synaptophysin. Upon overexpression of synaptophysin in PC12 cells, it is localized in rapidly sedimenting membranes and practically is not delivered to the SLMVs. On the contrary, the efficiency of the SLMV targeting of exogenously expressed cellugyrin and synaptogyrin is high. Moreover, expression of cellugyrin (or synaptogyrin) in PC12 cells dramatically and specifically increases SLMV targeting of endogenous synaptophysin. Finally, we utilized the SLMV purification scheme on a series of non-neuroendocrine cell types including the mouse fibroblast cell line 3T3-L1, the Chinese hamster ovary cell line CHO-K1, and the monkey kidney epithelial cell line COS7 and found that a cellugyrin-positive microvesicular compartment was present in all cell types tested. We suggest that synaptic vesicles have evolved from cellugyrin-positive ubiquitous microvesicles and that neuroendocrine SLMVs represent a step along that pathway of evolution. (+info)
Cellugyrin induces biogenesis of synaptic-like microvesicles in PC12 cells.
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The four-transmembrane domain proteins synaptophysin and synaptogyrin represent the major constituents of synaptic vesicles. Our previous studies in PC12 cells demonstrated that synaptogyrin or its nonneuronal paralog cellugyrin targets efficiently to synaptic-like microvesicles (SLMVs) and dramatically increases the synaptophysin content of SLMVs (Belfort, G. M., and Kandror, K. V. (2003) J. Biol. Chem. 278, 47971-47978). Here, we explored the mechanism of these phenomena and found that ectopic expression of cellugyrin increases the number of SLMVs in PC12 cells. Mutagenesis studies revealed that cellugyrin's hydrophilic cytoplasmic domains are not involved in vesicle biogenesis, whereas small conserved hydrophobic hairpins in the first luminal loop and the carboxyl terminus of cellugyrin were found to be critical for the formation of SLMVs. In addition, the length but not the primary sequence of the second luminal loop was essential for SLMV biogenesis. We suggest that changing the length of this loop similar to disruption of the short hydrophobic hairpins alters the position of the vicinal transmembrane domains that may be crucial for protein function. (+info)
The luminal Vps10p domain of sortilin plays the predominant role in targeting to insulin-responsive Glut4-containing vesicles.
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In fat and skeletal muscle cells, insulin-responsive vesicles, or IRVs, deliver glucose transporter Glut4 and several associated proteins to the plasma membrane in response to hormonal stimulation. Although the protein composition of the IRVs is well studied, the mechanism of their formation is unknown. It is believed, however, that the cytoplasmic tails of the IRV component proteins carry targeting information to this compartment. To test this hypothesis, we have studied targeting of sortilin, one of the major IRV constituents. We have found that the reporter protein consisting of the cytoplasmic tail of sortilin and EGFP is co-localized with ectopically expressed Glut4 in the perinuclear compartment of undifferentiated 3T3-L1 cells that do not form insulin-responsive vesicles. Upon cell differentiation, this reporter protein does not enter the IRVs; moreover, it loses its perinuclear localization and becomes randomly distributed throughout the whole intracellular space. In contrast, the tagged luminal Vps10p domain of sortilin demonstrates partial co-localization with Glut4 in both undifferentiated and differentiated cells and is targeted to the IRVs upon cell differentiation. Using chemical cross-linking and the yeast two-hybrid system, we show that sortilin interacts with Glut4 and IRAP in the vesicular lumen. Our results suggest that luminal interactions between component proteins play an important role in the process of IRV biogenesis. (+info)