Fig. 1. The N-terminus of VAMP4 mediates recycling from the cell surface to the TGN. (A) EGFP-fusion protein expression constructs. The entire coding region of VAMP4 and VAMP5 was inserted into the pEGFP-N1 vector so that VAMP4 and VAMP5 are expressed as C-terminally EGFP-tagged proteins. The coding region of the N-terminal extension (residues 1-48) of VAMP4 was fused in-frame with the second residue of VAMP5 to create a construct for expressing V4nV5-EGFP (which consists of VAMP4 N-terminal extension fused to VAMP5-EGFP). NE, N-terminal extension; SNARE, SNARE domain; TM, transmembrane domain. NRK cells were then transfected with each of these constructs and pools of stably transfected cells were used in subsequent experiments. (B) NRK cells stably expressing VAMP4-EGFP (a-c), VAMP5-EGFP (d-f) and V4nV5-EGFP (g-i) were incubated at 37°C in the continuous presence of anti-EGFP antibody for 60 minutes. Panels a,d,g show the EGFP signals of these fusion proteins. Anti-EGFP antibody was detected by Cy3-conjugated goat anti-rabbit secondary antibody (b,e,h). The merged images are also shown (c,f,i). Bar, 10 µm.