The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3– and AP-1–bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3–positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3–deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.

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The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosynand VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction cosediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.The exocytosis of synaptic and dense core vesicles with the plasma membrane in neurons and neuroendocrine cells requires proteins of the SNARE 1 families. The vesicle-associated membrane protein, VAMP2, a v-or R-SNARE, forms a specific SNARE complex with the target membrane-associated t-or Q-SNAREs syntaxin 1 and SNAP-25, whose parallel orientation brings the two membranes in close enough proximity to fuse (1, 2). Structurally the SNARE complex is a four-helix bundle comprised of one coiled-coil-forming domain from each of syntaxin and VAMP and two from 4). The center of the bundle is made up of 15 hydrophobic layers from the "a" and "d" positions of the heptad repeats of these coiled-coilforming domains, whereas the central "ionic" layer is highly conserved and polar in nature, containing a glutamine residue in the three t-SNAREs and an arginine in the v-SNARE (3), hence the classification of v-and t-SNAREs as R-and QSNAREs, respectively (5). The parallel orientation and high stability of the SNARE bundle led to the proposal that its formation drives the mixing of the membrane bilayers by zippering up through the transmembrane domains of syntaxin and VAMP (1, 2), and there is some evidence to support this (6 -9). After the fusion event, the SNAREs are in a cis-complex in the same membrane and must be dissociated by the action of the ATPase NSF and ␣-SNAP, so that SNAREs can be regenerated for the next round of fusion (10 -13).In addition to mediating the fusion event, the SNARE proteins have also been implicated in the specificity of membrane fusion, the SNARE hypothesis stating that a particular v-SNARE on a transport vesicle should only form a specific complex with its cognate t-SNARE on the correct target membrane, thereby ensuring that the vesicles only fuse with the right compartment (14). Although this hypothesis was cast into doubt by the finding that soluble ...