Cellugyrin Induces Biogenesis of Synaptic-like Microvesicles in PC12 Cells

Abstract

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 synapticlike 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.Understanding the mechanisms by which a cell sequesters specific proteins in unique subcellular compartments is fundamental to understanding how the cell maintains distinct organelles. For the past 25 years, the PC12 neuroendocrine cell line has provided a convenient system for studying the biogenesis of synaptic like microvesicles (SLMVs) 1 (1). SLMVs were chosen as a model organelle, because, like neuronal synaptic vesicles, their small size (ϳ50 nm) restricts the number of proteins present in each vesicle. Based on the estimates of 3 ϫ 10 6 daltons of protein/synaptic vesicle and 50,000 daltons/average protein, ϳ60 proteins could be contained in a vesicle of this size (2).Two main methods have been used to study the biogenesis of SLMVs. Several groups have studied cell-free or perforated cell vesicle budding systems in order to identify cytosolic factors critical for vesicle budding. Others studying specific proteins that target to SLMVs have identified regions in their primary amino acid sequences that are required for targeting to that compartment.Two main pathways of SLMV biogenesis have emerged from in vitro studies. First, vesicles can bud from endosomes in a brefeldin A-sensitive mechanism that requires the AP3 adaptor protein and the ARF1 and Rab4 GTPases (3-6). By the second pathway, SLMVs can bud from the plasma membrane in a brefeldin A-insensitive mechanism that involves the AP2 adapter, clathrin, and dynamin (7). Further work with perforated PC12 cells demonstrated that endophilin I, a dynaminbinding protein, facilitates SLMV budding, probably by modifying the membrane lipid composition (8).Two categories of proteins are present in SLMVs in PC12 cells: neuronal synaptic vesicle proteins and other proteins that are not found in brain synaptic vesicles. Of those proteins belonging to the former category, synaptophysin, synaptobrevin/VAMPII, the vesic...

The C-terminus of GLUT4 targets the transporter to the perinuclear compartment but not to the insulin-responsive vesicles

“…Extracts of transfected adipocytes were incubated with an excess of the anti-Myc antibody (or non-specific IgG) together with nanogold-conjugated goat anti-mouse Fab fragments. During the incubation period, the heavy antibody–Fab–nanogold complex bound to the cytoplasmic Myc epitope of the target proteins, and ‘decorated’ and ‘non-decorated’ vesicles were then analysed by sucrose-gradient centrifugation (see also [23]).…”

“…Unlike IRVs that compartmentalize most of the intracellular GLUT4, cellugyrin-containing vesicles are completely resistant to insulin stimulation and cellugyrin is not translocated to the plasma membrane in response to insulin ([21,22] and the present study). Last, but not least, the C-terminus of cellugyrin does not carry any targeting information [23], which is essential for the interpretation of results. We have found that the C-terminus of GLUT4 is required for protein localization in the perinuclear compartment, but is not sufficient for the efficient targeting to the IRVs.…”

Abnormal Synaptic Vesicle Biogenesis inDrosophila SynaptogyrinMutants

“…Synaptophysin binds cholesterol and promotes the formation of highly curved membranes (Thiele et al, 2000). Overexpression of cellugyrin, a non-neuronal paralog of synaptogyrin, induces the formation of synaptic-like microvesicles (SLMVs) in neuroendocrine cells (Belfort et al, 2005). Synaptophysin may also participate in synaptic vesicle recycling through a Ca 2+ -dependent interaction with dynamin, as the inhibition of this interaction results in a smaller synaptic vesicle pool following high-frequency stimulation (Daly et al, 2000; Daly and Ziff, 2002).…”

Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans

“…This notion is supported by the multiple interactions of TVPs with lipids, notably cholesterol (8) or various components of the recycling machinery including soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) (9-11), dynamin (12, 13), adaptor proteins (14), and eps15 homology (EH)-domain proteins (15). In addition, it has been postulated that TVPs are directly responsible for microvesicle formation (8,16,17) and are involved in fusion pore formation (18-21).…”