Sorting signals have been identified in VMAT2 (A) VAChT (B) and VGLUT1 (C). All three proteins are likely to contain twelve hydrophobic domains (5, 112, 113, 151), but only ten may completely cross the membrane in VGLUT1 (152). As in Fig. 1, signals and pathways for sorting to SVs and SLMVs are colored purple; those relevant to LVs are orange. Sorting signals in VGAT/VIAAT are not known and the prediction of ten transmembrane domains (153, 154) has not been experimentally tested. A) In VMAT2 (and VAChT) a large lumenal loop between transmembrane domains 1 and 2 is glycosylated (orange “Y”). Glycosylation at this site may facilitate sorting of VMAT2 to LVs (see text). All other known sorting signals are encoded in the C-terminal, cytoplasmic domains of the vesicular transporters. For VMAT2, this includes a dileucine motif (IL) that is required for endocytosis and sorting to SLMVs in neuroendocrine cells (95). The endocytosis motif is embedded in a larger, extended dileucine motif that includes upstream glutamate residues (EE). The upstream glutamate residues are required for sorting into the regulated secretory pathway and onto LVs (44, 102). The initial step for sorting onto LVs is likely to occur at the TGN and involve sorting away from constitutive secretory vesicles (CS) and onto immature LVs (im. LV). A phosphorylated acidic cluster at the extreme C-terminus of VMAT2 (DDEE[P]SE[P]SD) helps retain VMAT2 in the LV as it matures. B) VAChT also contains a dileucine motif (LL) required for endocytosis and sorting to SLMVs (95, 96, 110). However, a novel tyrosine based motif YNYY may direct these trafficking events under some circumstances (111). A serine upstream of the dileucine motif ([P]S) in VAChT undergoes phosphorylation by PKC (102, 129). Substitution of an acidic residue to mimic phosphorylation drives a portion of VAChT into the regulated secretory pathway and on to LVs in PC12 cells (102). D) Endocytosis and recycling of VGLUT1 to SVs in hippocampal neurons requires a dileucine-like motif (FV). Under some circumstances, a polyproline motif that binds endophilin also contributes to VGLUT1 recycling to SVs (112). A distinct pathway that does not require the polyproline motif allows compensatory recycling of VGLUT, and requires the AP3 adaptor complex (112)(not shown).

Trafficking Pathways Proposed for Vesicular Neurotransmitter Transporters. A) Secretory vesicle subtypes. Vesicular transporters localize to different types of secretory vesicles in neurons. These include synaptic vesicles (labeled SV in the figure), and large dense core vesicles (LDCVs, labeled LV in the figure). LVs, but not synaptic vesicles, co-release neuromodulatory peptides, Both SVs and LVs undergo calcium-mediated exocytosis (red arrows) but differ in the site and mode of release. SVs undergo regulated release in nerve terminals and more specifically, at the active zone, a discrete presynaptic region located opposite to post-synaptic receptors. Unlike SVs, LVs are found in both the nerve terminal as well as the cell body and dendrites, and do not closely appose the plasma membrane. The release of neurotransmitter from LVs activates receptors outside of the synapse (labeled “Extra synaptic receptors” in panel A) rather than postsynaptic receptors. The release of neurotransmitter from LVs can also activate somatodendritic autoreceptors, which are found on both the nerve terminal and somatodendritic membranes of most if not all neurons, and provide negative feedback to limit excess neurotransmitter release.
B) Trafficking to secretory vesicle subtypes. Vesicular transporters traffic to SVs and LVs via different routes. For sorting to SVs (purple arrows), proteins are thought the exit the trans Golgi network (TGN) in constitutive secretory (CS) vesicles, and are likely to undergo an endocytic step to generate a mature SV. In contrast, proteins destined for LVs sort into the regulated secretory pathway at the TGN (orange arrows). Immature LVs (im. LVs) are thought to mature via AP1- and PACS-1 dependent removal of some proteins, shown here as a budding event.
C) Synaptic vesicle trafficking at the synapse. The generation of mature SVs as well as SV recycling at the nerve terminal may occur via several mechanisms. Following fusion with the plasma membrane, transporters on constitutive secretory (CS) vesicles may be sorted to a mature SV directly from the plasma membrane using clathrin and the adaptor complex AP2. Alternatively, they may sort to SVs via a two-step pathway involving AP3 and budding from an early endosomal intermediate (EE). Following exocytosis, SVs and vesicular transporters may recycle via either of these pathways. It is also possible that SVs recycle using a kiss-and-run mechanism (not shown).
D) Alternative models of transporter trafficking. Alternative models have been proposed for the sorting of some vesicular transporters to SLMVs in neuroendocrine cells (64, 65) (pathway labeled “PC12?”), and possibly to SVs in neurons (pathway labeled “Neurons?”). In one alternative model, proteins such as VAChT that are removed from immature LVs are sorted to an early endosome where they are sorted into SLMVs. Vesicles that leave immature LVs may represent a “constitutive-like” secretory pathway as originally proposed for secretory granules in pancreatic islet cells (63, 150). Trafficking of constitutive-like vesicles to the nerve terminal might allow sorting to mature SVs via endocytosis.