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First published online 27 February 2007
doi: 10.1242/jcs.03387

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

Institute of Molecular and Cell Biology, Proteos Building, 61 Biopolis Drive, 138673 Singapore

View larger version (39K): [in this window] [in a new window]   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.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Involvement of vesicular intermediates in VAMP4-EGFP recycling. NRK cells expressing VAMP4-EGFP (upper panels) or V4nV5-EGFP (lower panels) were incubated with anti-EGFP antibody at 4°C for 1 hour to allow binding to surface fusion proteins. After a brief washing, cells were incubated at 37°C in the absence of antibody for the indicated periods of time to follow the trafficking of fusion protein-bound antibody. EGFP-fusion protein fluoresces green, whereas anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). As revealed, recycling of VAMP4-EGFP and V4nV5-EGFP involves peripheral and peri-Golgi vesicular intermediates before being concentrated in the Golgi complex. Bars, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Inhibition of internalization of VAMP4-EGFP by treatments blocking clathrin-mediated endocytosis. (A) After a hypotonic shock, NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP in the absence (a-f) or presence (g-l) of K+ for 30 (a-c and g-i) or 60 minutes (d-f and j-l). EGFP-fusion protein fluoresces in green, while anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). The merged images are also shown (c,f,i and l). (B) After hypertonic treatment, NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP in the presence (a-f) or absence (g-l) of 0.45 M sucrose for 30 (a-c and g-i) or 60 minutes (d-f and j-l). EGFP-fusion protein fluoresces in green and anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). The merged images are also shown (c,f,i,l). Bars, 10 µm.

View larger version (113K): [in this window] [in a new window]   Fig. 4. VAMP4-EGFP recycles through the sorting and recycling endosomes labeled by EEA1 and transferrin receptor (TfR). (A) NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP antibody at 4°C for 1 hour to label the surface pool of VAMP4-EGFP. Cells were then incubated at 37°C in the absence of antibody for 2-40 minutes. Endogenous EEA1 is shown in green, while anti-EGFP antibody is shown in red. The merged images are also shown. (B) NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP antibody and anti-TfR at 4°C for 1 hour. After washing, cells were then incubated at 37°C in the absence of the antibody for 2-60 minutes. Anti-TfR is green and anti-EGFP antibody is red. The merged images are also shown. Bars, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 5. VAMP4-EGFP recycling was arrested at the peri-Golgi recycling endosomes (REs) at 18°C. Cells expressing VAMP4-EGFP were incubated at 37°C (a-c) or at 18°C (d-i) with either anti-EGFP alone (g-i) or both anti-EGFP and Alexa Fluor 647-conjugated transferrin (Tf-AF647) (a-f) for 1 hour. Anti-EGFP is red (b,e,h) while Tf-AF647 (a,d) or endogenous LBPA (revealed by antibody labeling) (g) is green. The merged images are also shown (c,f,i). Panel f1 shows the enlarged image of a part of panel f. As a control, NRK cells were incubated at 18°C with FITC-conjugated transferrin (Tf-FITC) for 1 hour (j-l). Cells were then analyzed to view the internalized transferrin (j) and endogenous GM130 as revealed by antibody labeling (k). The merged image is shown in panel l. Panel l1 shows the enlarged image of a part of panel l, indicating that the peri-Golgi RE are segregated from the Golgi complex. Panels m-o show another control experiment where cells expressing VAMP4-EGFP were incubated at 18°C with Tf-AF647 (green) and anti-EGFP (red) for 1 hour. Cells were then incubated in DMEM at 37°C for an additional 30 minutes before being fixed for viewing. Bar, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Nocodazole does not affect VAMP4-EGFP recycling. After a 30-minute pretreatment with 33 µM nocodazole (a-d) or 0.1% DMSO (e-h), VAMP4-EGFP-expressing cells were incubated with rabbit anti-EGFP antibody for 30 minutes at 37°C in the continuous presence of nocodazole (a-d) or DMSO (e-h). Cells were then processed to reveal EGFP (green), anti-EGFP signals (red) and endogenous GS15 (blue). The merged images are also shown. Bar, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Arrest of VAMP4-EGFP recycling in peri-Golgi REs when vacuolar ATPase is inhibited by BFLA1 or conA. (A) NRK (j-o) or VAMP4-EGFP-expressing NRK (a-i) cells were pre-incubated with DMEM alone (a-c), in the presence of 50 nM BFLA1 (d-f,j-l), or in the presence of 100 nM conA (g-i,m-o) for 30 minutes. Cells were then incubated with anti-EGFP and Tf-AF647 (a-i) or Tf-FITC alone (j-o) for 30 minutes in the absence or presence of the drug as indicated. Fluorescent dye-conjugated Tf is shown in green, internalized anti-EGFP (b,e,h) or endogenous GM130 (k,n) is shown in red. Panels l1 and o1 are enlarged images of the indicated portion of the merged images, l and o, respectively. (B) VAMP4-EGFP-expressing NRK cells were pre-incubated with either 50 nM BFLA1 (a-c) or 100 nM conA (d-f) for 30 minutes. Cells were then incubated with anti-EGFP antibody and Tf-AF647 in the continuous presence of either drug for an additional 30 minutes, washed and subjected to a further 30-minute incubation in fresh DMEM, without the drug at 37°C. Tf-AF647 signal is green and anti-EGFP signal is red (see above). Bars, 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 8. The TGN targeting signal of VAMP4 plays a role in VAMP4-EGFP recycling. (A) The schematic illustration of expression constructs of VAMP4-EGFP and its mutants. Site-directed mutations were created in the context of VAMP4-EGFP. VAMP4-EGFP/LL-AA has the di-Leu motif (residues 25-26) replaced by two Ala residues. The first acidic cluster EDD (residues 27-29) was mutated into three Ala residues in VAMP4-EGFP/EDD-3A. VAMP4-EGFP/DEEED-5A has the distal acidic cluster (DEEED, residues 31–35) replaced by a stretch of five Ala residues. The two Phe residues at positions 36-37 were replaced by Ala residues in VAMP4-EGFP/FF-AA. NE, N-terminal extension; SNARE, SNARE domain; TM, transmembrane domain. These constructs were each transfected into NRK cells and pools of stable transfectants were selected and expanded for the experiments. (B) NRK cells expressing VAMP4-EGFP (a-c), VAMP4-EGFP/LL-AA (panels d-f), VAMP4-EGFP/EDD-3A (panels g-i), VAMP4-EGFP/DEEED-5A (panels j-l) and VAMP4-EGFP/FF-AA (panels m-o) were incubated at 4°C with rabbit anti-EGFP antibody in cold DMEM for 1 hour. After a brief wash, cells were then incubated in fresh DMEM at 37°C for 15 minutes. Panels a,d,g,j,m show the EGFP signal of these fusion proteins. Anti-EGFP antibody was detected by Cy3-conjugated goat anti-rabbit secondary antibody (b,e,h,k,n). The merged images are also shown (c,f,i,l,o). (C) Cells from B were washed twice in cold acidic washing buffer to strip off antibody remaining bound on the surface before being fixed for immunofluorescence microscopy. Panels a, d, g, j and m show the EGFP signal of these fusion proteins. Anti-EGFP antibody was detected by Cy3-conjugated goat anti-rabbit secondary antibody (panels b,e,h,k,n). The merged images are also shown (panels c,f,i,l,o). Bars, 10 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 9. A schematic model depicting the recycling pathway of VAMP4. VAMP4-EGFP internalized from the surface by clathrin-mediated endocytosis is firstly delivered to the sorting endosomes (SE) labeled with EEA1 and TfR and then transported to the peri-Golgi REs labeled with TfR and revealed by its accumulation there when cells were incubated at 18°C or treated with BFLA1 or conA before being delivered to the TGN. The di-Leu motif of its TGN-targeting signal is necessary for the internalization from the surface whereas the distal acidic cluster is involved in efficient delivery from the endosome to the TGN. The strong correlation of the signal responsible for TGN targeting and recycling indicates that this recycling pathway contributes to the steady-state enrichment of VAMP4 in the TGN. The elucidation of this recycling pathway is in line with the function of VAMP4 as a v-SNARE participating in trafficking from the REs to the TGN.