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i-Vamp, and Vamp8/endobrevin) that are distributed in various post-Golgi structures. Vamp4 and Vamp5 are distributed predominantly in the trans-Golgi network (TGN) and the plasma membrane, respectively. When C-terminally tagged with enhanced green fluorescent protein, the majority of Vamp4 and Vamp5 is correctly targeted to the TGN and plasma membrane, respectively. Swapping the N-terminal cytoplasmic region and the C-terminal membrane anchor domain between Vamp4 and Vamp5 demonstrates that the N-terminal cytoplasmic region of these two SNAREs contains the correct subcellular targeting information. As compared with Vamp5, Vamp4 contains an N-terminal extension of 51 residues. Appending this 51-residue N-terminal extension onto the N terminus of Vamp5 results in targeting of the chimeric protein to the TGN, suggesting that this N-terminal extension of Vamp4 contains a dominant and autonomous targeting signal for the TGN. Analysis of deletion mutants of this N-terminal region suggests that this TGN-targeting signal is encompassed within a smaller region consisting of a di-Leu motif followed by two acidic clusters. The essential role of the di-Leu motif and the second acidic cluster was then established by site-directed mutagenesis.