Dileucine motif is sufficient for internalization and synaptic vesicle targeting of vesicular acetylcholine transporter.
Efficient cholinergic transmission requires accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs). However, the signals that regulate this vesicular targeting are not well characterized. Although previous studies suggest that the C-terminus of the transporter is required for its SV targeting, it is not clear whether this region is sufficient for this process. Furthermore, a synaptic vesicle-targeting motif (SVTM) within this sequence remains to be identified. Here we use a chimeric protein, TacA, between an unrelated plasma membrane protein, Tac, and the C-terminus of VAChT to demonstrate the sufficiency of the C-terminus for targeting to synaptic vesicle-like vesicles (SVLVs) in PC12 cells. TacA shows colocalization and cosedimentation with the SV marker synaptophysin. Deletion mutation analysis of TacA demonstrates that a short, dileucine motif-containing sequence is required and sufficient to direct this targeting. Dialanine mutation analysis within this sequence suggests indistinguishable signals for both internalization and SV sorting. Using additional chimeras as controls, we confirm the specificity of this region for SVLVs targeting. Therefore, we suggest that the dileucine-containing motif is sufficient as a dual signal for both internalization and SV targeting during VAChT trafficking.
Colgan
Lesley
L
Department of Neurology, University of Pittsburgh School of Medicine, S512 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
Liu
Hao
H
Huang
Steven Y
SY
Liu
Yong-Jian
YJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D004151:Dipeptides / Q000235:genetics / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016716:PC12 Cells; D010446:Peptide Fragments / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011990:Receptors, Transferrin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins / Q000235:genetics / Q000378:metabolism*; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism
D004151:Dipeptides; D051275:Glucose Transporter Type 4; D053645:Interleukin-2 Receptor alpha Subunit; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C495692:SLC2A4 protein, human; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; C038361:leucylleucine