The Cytoplasmic Tail of the Vesicular Acetylcholine Transporter Contains a Synaptic Vesicle Targeting Signal

Abstract

The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles.The organelles used for regulated biogenic amine and acetylcholine (ACh) 1 secretion in neurons and endocrine cells are the large and small dense core vesicles (DCV) and small synaptic vesicles, respectively. The biogenesis and fate of these organelles and targeting of various proteins to them differ in several ways (1-6). LDCVs (approximately 200 nm) are the product of the trans-Golgi network as they contain soluble glycoproteins, such as the chromogranins, and various neuropeptides. Following maturation and subsequent exocytosis at the plasma membrane, components of the LDCV membrane must recycle to the Golgi complex to replenish their soluble neurohormone content and re-enter the regulated secretory pathway. In contrast, synaptic vesicle proteins, such as synaptophysin (p38), appear to travel to the plasma membrane via the constitutive secretory pathway and are then endocytosed and sorted in endosomes, from which synaptic vesicles (40 nm) bud. Exocytosis/endocytosis and recycling of cholinergic synaptic vesicles takes place at the presynaptic nerve terminal, while neuropeptide-and monoamine-containing LDCVs, located throughout the cell, may release nonsynaptically (7-10).Cloning and characterization of the family of vesicular transporters for biogenic amines and ACh has provided new tools for the study of the biogenesis of these regulated secretory organelles (11, 12). The examination of the subcellular localization of these proteins indicates that they are associated with distinct vesicle populations in neurons...

The Vesicular Acetylcholine Transporter Interacts with Clathrin-associated Adaptor Complexes AP-1 and AP-2

“…An alternative pathway for VAChT trafficking, suggested by Tao-Cheng and Eiden (54) and by Varoque and Erickson (35), utilizes the regulated secretory sorting pathway, as do VMATs, instead of the constitutive secretory pathway, Fig. 10B.…”

“…The cytoplasmic tail of a number of membrane proteins has been shown to contain information for targeting to various intracellular destinations (32)(33)(34)(35). The VAChT cytoplasmic carboxyl tail fits into this group as it has been shown to be involved in the targeting of this transporter to synaptic vesicles (35).…”

“…The VAChT cytoplasmic carboxyl tail fits into this group as it has been shown to be involved in the targeting of this transporter to synaptic vesicles (35). This segment of VAChT contains two putative sorting motifs; a dileucine motif preceded by a phosphorylation site and two possible tyrosine-based sorting motifs, Fig.…”

“…Although PC12 cells express endogenous VAChT mRNA as detected by PCR, VAChT protein expression is too low to be detected by immunoprecipitation and Western blot analysis. Therefore, as done in other studies (4,35) full-length VAChT was stably transfected into PC12 cells to produce PC12 VAChT . In general cargo protein-adaptor protein complex interactions are transient and unstable.…”

A Conserved Mechanism of Synaptogyrin Localization

“…Several studies have identified domains and motifs necessary for correct localization of SV proteins. Targeting and sorting signals have been identified in synaptophysin (Linstedt and Kelly, 1991), synaptobrevin (Grote et al, 1995;Grote and Kelly, 1996), synaptotagmin (Blagoveshchenskaya et al, 1999;Krasnov and Enikolopov, 2000), and vesicular neurotransmitter transporters (Tan et al, 1998;Varoqui and Erickson, 1998). However, no common motif that could serve as a universal targeting signal has been found by experimental studies or by direct comparison of the primary sequences of SV proteins.…”