Intracellular trafficking of vesicular monoamine transporters

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family and package acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or synaptic vesicles, respectively. When expressed in PC12 cells, vesicular monoamine transporter 2 (VMAT2) localizes to LDCV, whereas VAChT was found mainly on synaptic-like microvesicles (SLMVs). Previous studies have shown that the cytoplasmic C-terminus of VAChT contains the synaptic vesicle targeting signals. However, the targeting signals for VMAT have not been fully elucidated. To identify the possible trafficking signals targeting VMAT2 to LDCV, chimeras of VMAT2 and VAChT were expressed in PC12 cells, and their localization assessed by linear sucrose gradient fractionation. Chimeras of VAChT containing either the cytoplasmic C-terminus of VMAT2 or the N-terminal region of VMAT2 do not traffic to LDCV. However a chimera containing both the cytoplasmic C-terminus of VMAT2 and the N-terminal region, or the N-linked glycosylated loop and the cytoplasmic C-tail, directed the chimera to LDCV. Thus, although only the cytoplasmic C-terminus of VAChT is required for its SLMV targeting, both the N-terminal domain containing N-linked glycosylation, and the cytoplasmic C-terminus of VMAT2 are required for LDCV targeting. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, caused VMAT2 to localize to synaptic-like microvesicles in PC12 cells. Thus the pattern of N-linked glycosylation serves as part of the trafficking signals for VMAT2. These data indicate that the location of the trafficking signals within these highly related transporters is surprisingly diverse.; Protein kinase A (PKA) appears to play a critical role in the trafficking of VMAT in PC 12 cells. When VMAT2 was expressed in PKA deficient PC12 cells, it mislocalized to SLMVs. Co-expression of the catalytic subunit of PKA partially restored trafficking of VMAT2 to LDCV and treatment of wild type PC12 cells with the PKA inhibitor, H89, increased VMAT2 sorting to SLMVs. Interestingly, the VMAT2 in LDCV had a higher molecular weight; a difference that appeared to be due to differential N-linked glycosylation. In vitro phosphorylation studies showed that PKA does not phosphorylate VMAT2, suggesting that PKA's in vivo role in VMAT2 sorting is not through direct phosphorylation. Possible mechanisms for VMAT sorting and PKA's role in the process are discussed.