spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 19 April 2005
doi: 10.1242/jcs.02315

Journal of Cell Science 118, 1885-1897 (2005)
Published by The Company of Biologists 2005
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, P. J. C.
Right arrow Articles by Numata, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, P. J. C.
Right arrow Articles by Numata, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7

Paulo J. C. Lin1, Warren P. Williams1, Yvonne Luu1, Robert S. Molday1, John Orlowski2 and Masayuki Numata1,*

1 Department of Biochemistry and Molecular Biology, The University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC V6T 1Z4, Canada
2 Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada



View larger version (25K):

[in a new window]
  		Fig. 1. SCAMPs interact with NHE7. (A) Schematic representation of SCAMP1, 2 and 5 showing the Asn-Pro-Phe (NPF) repeat and a highly conserved region (underlined) containing a Pro-rich (P rich) motif and four transmembrane (TM) segments. (B) CHO cells were transiently transfected with HA-tagged NHE7 or NHE6 and myc-tagged SCAMP1, 2 or 5. Cells were lysed in 0.5% NP40/PBS for 30 minutes on ice and lysates were cleared for 20 minutes at 16,000 g at 4°C. Cell lysates (Lys) were then immunoprecipitated with mouse anti-HA antibody (HA) or pre-immune serum (con) and bound SCAMPs were detected on western blots probed with rabbit anti-myc antibody. 5% of the total lysate was loaded. (C) GST and GST fused with NHE7 C-terminal tail (GST-NHE7 [542-725]) were expressed in E. coli and purified by incubation with glutathione-conjugated sepharose beads. 2 µg immobilized GST or GST-NHE7 C-terminal fusion protein was incubated with PC12 cell lysate, and any SCAMPs bound to the GST fusion protein were detected by western blotting.

 


View larger version (14K):

[in a new window]
  		Fig. 2. Identification of SCAMP2 binding domain within NHE7. GST alone and GST fused to various segments of NHE7 (residues 542-725, 615-725, 666-725, 542-614 and 542-665, as illustrated below) were purified by glutathione-sepharose beads and subjected to GST pull-down. The radiolabeled SCAMP2 protein was incubated with 2 µg purified GST or GST fusion proteins immobilized on glutathione sepharose beads. After extensive washing, bound SCAMP2 was eluted with SDS sample buffer, resolved by SDS-PAGE, and visualized using a PhosphorImager.

 


View larger version (47K):

[in a new window]
  		Fig. 3. Co-immunoprecipitation of SCAMP2myc deletion mutants and NHE7HA. CHO cells were transiently co-transfected with myc-tagged SCAMP2 deletion mutants as illustrated above the blots, and HA-tagged NHE7. Pre-cleared lysates (Lys) were immunoprecipitated with an anti-HA antibody (HA) or mouse pre-immune serum (con) and bound SCAMP2 was detected on western blot by an anti-myc antibody. 5% of the lysate was run for each sample.

 


View larger version (37K):

[in a new window]
  		Fig. 4. Dose-dependent binding of SCAMP2[201-215] to NHE7. (A) Predicted membrane topology of SCAMP2. (B) Increasing amounts of GST-SCAMP2[201-215] immobilized to glutathione beads were incubated with radiolabelled NHE7 C-terminus and the bound NHE7 was visualized using a PhosphorImager. 4 µg GST was used as a control.

 


View larger version (42K):

[in a new window]
  		Fig. 5. Endogenous SCAMPs bind and largely colocalize with NHE7. (A) Control PC12 cells (PC12) or PC12 cells stably expressing 1D4-tagged NHE7 (NHE7/PC12) were immunoprecipitated (IP) with the 1D4 antibody conjugated to sepharose beads. Co-immunoprecipitated SCAMP1, 2, or 5 was detected by western blot probed with anti-SCAMP antibodies. (B) Fixed NHE7/PC12 cells on glass coverslips were double stained with anti-SCAMP1, SCAMP2 or SCAMP5 rabbit antibody with anti-1D4 mouse antibody. SCAMP1 (a), SCAMP2 (d) and SCAMP5 (g) were visualized with Alexa 488-conjugated goat anti-rabbit IgG (green) and the corresponding NHE7 by Alexa 568-conjugated anti-mouse IgG (red fluorescence) (b, e and h respectively). Yellow signals in merged images (c, f and i respectively) correspond to colocalized proteins. Bar, 10 µm.

 


View larger version (114K):

[in a new window]
  		Fig. 6. NHE7 and SCAMPs co-fractionate by sucrose equilibrium density centrifugation. Homogenate was prepared from PC12 cells stably transfected with 1D4-tagged NHE7 and analyzed by sucrose equilibrium density centrifugation. Fifteen fractions were taken from the top and an equal volume of each fraction was analyzed on a western blot probed with anti-1D4 and different antibodies that recognize endogenous proteins. The following organellar markers were used: GM130 (cis/medial-Golgi), syntaxin 6 (Stx6; TGN/TGN-derived vesicles), syntaxin 13 (Stx13; recycling endosome), Rab 11 (recycling endosome), transferrin receptor (TfR; recycling endosome), ß-COP (cis-Golgi), synaptophysin (Syp; synaptic-like microvesicles), PDI (ER) and EEA1 (early endosomes). Note that NHE7 and SCAMPs showed similar peaks in fractions 7, 8 and 9, but SCAMPs have broader distribution (fractions 5 and 6). In SCAMP2, a less prominent 35 kDa band (labelled with asterisks) was observed in addition to the expected 38 kDa band. This minor band probably represents a non-specific reaction, but could also reflect a proteolytically cleaved form.

 


View larger version (40K):

[in a new window]
  		Fig. 7. NHE7 and SCAMPs colocalize with syntaxin 6 and transferrin receptor. Endogenous SCAMP1, -2, and -5 were visualized in PC12 cells using anti-SCAMP polyclonal rabbit antibodies. To determine intracellular localization of NHE7, PC12 cells were transiently transfected with myc-tagged NHE7 and an anti-myc polyclonal rabbit antibody was utilized. Anti-syntaxin 6 (TGN marker) and transferrin receptor (recycling endosomal marker) monoclonal mouse antibodies were used as organellar markers. NHE7 or SCAMPs were visualized with Alexa 488-conjugated goat anti-rabbit IgG (green) and syntaxin 6 or transferrin receptor was visualized with Alexa 568-conjugated anti-mouse IgG (red). NHE7 exhibited the best colocalization with syntaxin 6, whereas partial colocalization with transferrin receptor was also observed. SCAMPs showed close association with both syntaxin 6 and transferrin receptor, suggesting a more widespread distribution.

 


View larger version (44K):

[in a new window]
  		Fig. 8. Expression of SCAMP2{Delta}184-208 shows scattered vesicular appearance with NHE7 and full-length SCAMP. (A) CHO cells were transfected with myc-tagged {Delta}184-208 or full-length SCAMP2 and 1D4-tagged NHE7 (NHE7) and viewed by immunofluorescence microscopy. SCAMP2/{Delta}184-208 had a more scattered vesicular distribution than full-length wild-type SCAMP2. Co-transfected NHE7 was redistributed to scattered vesicular structures mostly in the same compartment as SCAMP2/{Delta}184-208. (B) CHO cells were simultaneously transfected with myc-tagged SCAMP2 deletion mutants and full-length HA-tagged SCAMP2, and their intracellular localization was visualized by immunofluorescence confocal microscopy. Expression of SCAMP2/{Delta}184-208, but not other mutants, redistributed full-length SCAMP2 to the same scattered vesicular structure. (C) CHO cells were transiently co-transfected with full-length HA-tagged SCAMP2 and myc-tagged SCAMP2/{Delta}184-208 or SCAMP2/{Delta}151-174. Cell lysates (Lys) were immunoprecipitated with a mouse anti-HA antibody (HA) or pre-immune serum (con) and co-precipitated SCAMP2/{Delta}184-208 and SCAMP2/{Delta}151-174 were detected in a western blot by rabbit anti-myc antibody. 5% of total cell lysate was loaded. Bar, 10 µm.

 


View larger version (100K):

[in a new window]
  		Fig. 9. SCAMP2/{Delta}184-208 colocalizes with the transferrin receptor at the peripheral region of cells. PC12 cells were transiently transfected with myc-tagged SCAMP2/{Delta}184-208 and its intracellular localization was analyzed by double-labelled immunofluorescence confocal microscopy with different organellar markers. (A) SCAMP2/{Delta}184-208 (green) colocalized with transferrin receptor (TfR, red) at the peripheral region of cells. (B) Neither syntaxin 6 (Stx6, red) nor GM130 (red) showed significant association with SCAMP2/{Delta}184-208 (green). Note that the expression of SCAMP2/{Delta}184-208 did not appreciably alter localization of these markers (see Fig. 4). Bar, 10 µm.

 


View larger version (49K):

[in a new window]
  		Fig. 10. The TM2-TM3 region is sufficient for NHE7 interaction. (A) CHO cells were transiently co-transfected with HA-tagged NHE7 and GFP-tagged SCAMP2 TM2-TM3 (GFP-TM2-3) or GFP control. Cell lysates (Lys) were immunoprecipitated with anti-HA antibody (IP) and bound GFP fusion proteins were analyzed on western blot. (B) Purified GST fusion protein of NHE7 C-terminus or GST was incubated with radiolabelled GFP-TM2-3 and bound protein was detected using a PhosphorImager. (C) CHO cells were simultaneously transfected with GFP-TM2-3 or GFP alone and NHE7HA and their intracellular localization was visualized by green and red fluorescence, respectively. (D) Homogenate isolated after transient transfection with GFP-TM2-3, GFP, SCAMP2myc or NHE7HA was analyzed by sucrose equilibrium density centrifugation. Bar, 3 µm.

 


View larger version (75K):

[in a new window]
  		Fig. 11. NHE7 C-terminus partially colocalizes with full-length SCAMP2. CHO cells were co-transfected with myc-tagged SCAMP2 (full-length or {Delta}184-208) and HA-tagged NHE7 C-terminus (G525-A725) and their localizations in the cell were visualized by immunofluorescence microscopy. NHE7 C-terminus showed significant colocalization with full-length SCAMP2, but only limited association with SCAMP2/{Delta}184-208. Representative cells with low-to-moderate expression levels are shown. Bar, 10 µm

 


View larger version (17K):

[in a new window]
  		Fig. 12. Proposed model showing that targeting of NHE7 from the recycling endosome to the TGN is facilitated by binding to SCAMPs. Expression of the SCAMP2/{Delta}184-208 mutant blocks the normal targeting by interfering with full-length SCAMP, whereas GFP-tagged SCAMP2 TM2-TM3 (GFP-TM2-3) blocks normal targeting of NHE7 by competitive inhibition with endogenous SCAMPs. EE, early endosomes; RE, recycling endosomes; PM, plasma membrane; TGN, trans-Golgi network.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2005