VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes.
VAMP4 is enriched in the trans-Golgi network (TGN) and functions in traffic from the early and recycling endosomes to the TGN, but its trafficking itinerary is unknown. Cells stably expressing TGN-enriched VAMP4 C-terminally-tagged with EGFP (VAMP4-EGFP) are able to internalize and transport EGFP antibody efficiently to the TGN, suggesting that VAMP4-EGFP cycles between the cell surface and the TGN. The N-terminal extension of VAMP4 endows a chimeric VAMP5 with the ability to cycle from the surface to the TGN. Detailed time-course analysis of EGFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments suggest that VAMP4-EGFP is endocytosed by clathrin-dependent pathways and is delivered to the sorting and then recycling endosomes. This is followed by a direct transport to the TGN, without going through the late endosome. The di-Leu motif of the TGN-targeting signal is important for internalization, whereas the acidic cluster is crucial for efficient delivery of internalized antibody from the endosome to the TGN. These results suggest that the TGN-targeting signal of VAMP4 mediates the efficient recycling of VAMP4 from the cell surface to the TGN via the sorting and recycling endosomes, thus conferring steady-state enrichment of VAMP4 at the TGN.
Tran
Ton Hoai Thi
TH
Institute of Molecular and Cell Biology, Proteos Building, 61 Biopolis Drive, 138673 Singapore.
Zeng
Qi
Q
Hong
Wanjin
W
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D002522:Chlorocebus aethiops; D004705:Endocytosis; D011992:Endosomes / Q000502:physiology*; D006056:Golgi Apparatus / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018942:Macrolides / Q000494:pharmacology; D009154:Mutation; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018942:Macrolides; D021382:Protein Sorting Signals; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C036978:concanamycin A