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Adaptor Protein Complex 3Adaptor Protein Complex 1Adaptor Protein Complex 2Adaptor Protein Complex 4Adaptor Protein Complex SubunitsAdaptor Protein Complex delta SubunitsAdaptor Protein Complex mu SubunitsAdaptor Proteins, Vesicular TransportAdaptor Protein Complex gamma SubunitsAdaptor Protein Complex beta SubunitsAdaptor Protein Complex alpha SubunitsAdaptor Proteins, Signal TransducingClathrinHuman CharacteristicsMonomeric Clathrin Assembly ProteinsGRB2 Adaptor ProteinShc Signaling Adaptor ProteinsAdaptor Protein Complex sigma SubunitsEndocytosisCoated VesiclesProtein Bindingtrans-Golgi NetworkClathrin-Coated VesiclesEndosomesMembrane ProteinsProtein TransportCarrier ProteinsMolecular Sequence DataAmino Acid SequenceTransport VesiclesPhosphoproteinsCoated Pits, Cell-MembraneGRB10 Adaptor ProteinGene Products, nefRecombinant Fusion Proteinssrc Homology DomainsMultiprotein ComplexesCell LineBrefeldin ASignal Transductionnef Gene Products, Human Immunodeficiency VirusMutationHeLa CellsProteinsCell MembraneLysosomesProto-Oncogene Proteins c-crkProtein Structure, TertiaryTransfectionPhosphorylationProtein Interaction MappingCRADD Signaling Adaptor ProteinMyeloid Differentiation Factor 88Two-Hybrid System TechniquesBinding SitesImmunoprecipitationTyrosineAmino Acid MotifsIntracellular Signaling Peptides and ProteinsNuclear ProteinsCOS CellsModels, BiologicalGRB7 Adaptor ProteinPrecipitin TestsDNA-Binding ProteinsSequence Homology, Amino AcidMice, KnockoutNerve Tissue ProteinsCytoskeletal ProteinsProtein Interaction Domains and MotifsSaccharomyces cerevisiae ProteinsJurkat CellsCrk-Associated Substrate ProteinHEK293 CellsOncogene ProteinsVesicular Transport ProteinsProto-Oncogene Proteins c-cblModels, MolecularCells, CulturedProtein-Tyrosine KinasesSaccharomyces cerevisiaeBase SequenceReceptors, Interleukin-1PhosphotyrosineMacromolecular SubstancesTranscription FactorsImmunoblottingEnzyme ActivationSon of Sevenless ProteinsRNA, Small InterferingCARD Signaling Adaptor ProteinsRecombinant ProteinsProto-Oncogene ProteinsDrosophila Proteinssrc-Family KinasesProtein SubunitsCytoplasmPhospholipase C gammaBlotting, WesternMembrane Glycoproteins

BLOC-1 complex deficiency alters the targeting of adaptor protein complex-3 cargoes. (57/155)

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.  (+info)

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3. (58/155)

Adaptor protein 3 (AP-3) is a vesicle-coat protein that forms a heterotetrameric complex. Two types of AP-3 subunits are found in mammalian cells. Ubiquitous AP-3 subunits are expressed in all tissues of the body, including the brain. In addition, there are neuronal AP-3 subunits that are thought to serve neuron-specific functions such as neurotransmitter release. In this study, we show that overexpression of neuronal AP-3 in mouse chromaffin cells results in a striking decrease in the neurotransmitter content of individual vesicles (quantal size), whereas deletion of all AP-3 produces a dramatic increase in quantal size; these changes were correlated with alterations in dense-core vesicle size. AP-3 appears to localize in the trans-Golgi network and possibly immature secretory vesicles, where it may be involved in the formation of neurosecretory vesicles.  (+info)

Rustling synaptic vesicle cargo after exocytosis. (59/155)

In this issue of Neuron, Voglmaier et al. provide new evidence that the retrieval of synaptic vesicle transporters after exocytosis proceeds along at least two different endocytic pathways. This work provides new insight into the mechanisms of sorting synaptic vesicle cargo at the cell surface.  (+info)

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. (60/155)

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.  (+info)

BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes. (61/155)

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.  (+info)

Identification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein. (62/155)

Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX phi-like adaptin-dependent sorting signal (Y31GTI34) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y31GTI34 (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y31GTI34 motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX phi-like sorting signals.  (+info)

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses. (63/155)

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.  (+info)

BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles. (64/155)

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.  (+info)