The cytoplasmic domain of Vamp4 and Vamp5 is responsible for their correct subcellular targeting: the N-terminal extenSion of VAMP4 contains a dominant autonomous targeting signal for the trans-Golgi network.
SNAREs represent a superfamily of proteins responsible for the last stage of docking and subsequent fusion in diverse intracellular membrane transport events. The Vamp subfamily of SNAREs contains 7 members (Vamp1, Vamp2, Vamp3/cellubrevin, Vamp4, Vamp5, Vamp7/Ti-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.
Zeng
Qi
Q
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore.
Tran
Thi Ton Hoai
TT
Tan
Hui-Xian
HX
Hong
Wanjin
W
D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D021601:trans-Golgi Network / Q000502:physiology*
D033942:Adaptor Proteins, Vesicular Transport; D017931:DNA Primers; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C476456:VAMP4 protein, human; C485545:VAMP5 protein, human; C476457:Vamp4 protein, mouse; C117769:Vamp5 protein, mouse; D049452:Green Fluorescent Proteins