VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes

J Cell Sci. 2007 Mar 15;120(Pt 6):1028-41. doi: 10.1242/jcs.03387. Epub 2007 Feb 27.

Abstract

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.

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / physiology*
  • Chlorocebus aethiops
  • Endocytosis
  • Endosomes / physiology*
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / physiology*
  • Green Fluorescent Proteins / genetics
  • Macrolides / pharmacology
  • Mutation
  • Protein Sorting Signals / physiology*
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Macrolides
  • Protein Sorting Signals
  • R-SNARE Proteins
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • concanamycin A