SNARE [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] proteins control the membrane-fusion events of eukaryotic membrane-trafficking pathways. Specific vesicular and target SNAREs operate in specific trafficking routes, but the degree of specificity of SNARE functions is still elusive. Apical fusion requires the polarized distribution at the apical surface of the t-SNARE syntaxin 3, and several v-SNAREs including TI-VAMP and VAMP8 operate at the apical plasma membrane in polarized epithelial cells. It is not known, however, whether specific v-SNAREs are involved in direct and indirect routes to the apical surface. Here, we used RNAi to assess the role of two tetanus-neurotoxin-insensitive v-SNAREs, TI-VAMP/VAMP7 and VAMP8, in the sorting of raft- and non-raft-associated apical markers that follow either a direct or a transcytotic delivery, respectively, in FRT or Caco2 cells. We show that TI-VAMP mediates the direct apical delivery of both raft- and non-raft-associated proteins. By contrast, sorting by means of the transcytotic pathway is not affected by TI-VAMP knockdown but does appear to be regulated by VAMP8. Together with the specific role of VAMP3 in basolateral transport, our results demonstrate a high degree of specificity in v-SNARE function in polarized cells.
The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.
Fast synaptic transmission is mediated by the secretion from nerve terminals of small nonpeptide chemical messengers, called neurotransmitters, which are stored in a specialized secretory organelle, the synaptic vesicle (De Camilli and Jahn 1990). Depolarization of the nerve terminal plasma membrane through an action potential leads to an influx of calcium ions into the cytosol that triggers the exocytosis of synaptic vesicles and the release of their contents. Rapidly after exocytosis, the synaptic vesicle membranes are internalized and reutilized for the formation of new synaptic vesicles that are reloaded with neurotransmitters from the cytosol (McPherson and De Camilli 1994). Several lines of evidence favor the view that this recycling process is mediated, at least in part (Fesce et al. 1994; Mundigl and De Camilli 1994), by the ubiquitous pathway of endocytosis that uses clathrin-coated vesicles. First, clathrin is highly concentrated in nerve terminals (Takei et al. 1995), and a morphological...
D-Serine is an astrocyte-derived regulator for N-methyl-D-aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D-serine. We report that D-serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D-serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+-ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D-serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D-serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D-serine.
The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster , most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider ( http://pim.hybrigenics.com ), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.
