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First published online 26 March 2003
doi: 10.1242/jcs.00415

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Identification of SAP97 as an intracellular binding partner of TACE

INSERM EPI 99-36, Laboratoire d'Hématologie, Faculté de Médecine, 27 Bd Jean Moulin, Marseilles 13385 Cedex 5, France

View larger version (48K): [in a new window]   Fig. 1. TACE interacts with the PDZ3 domain of SAP97. (A) Interaction analyzed by yeast two-hybrid system. TACE cytoplasmic tail and SAP97 mutant forms are expressed in yeast as fusion proteins with a GAL4 DNA-binding domain and a GAL4 activation domain, respectively (see Materials and Methods) and are schematically represented in the lower part of the figure. (B) Western blot showing that SAP97 mutant forms fused to the GAL4 activation domain are expressed in yeast. Detection was made using anti-SAP97 monoclonal antibody from C. Garner. (C) In vitro pull-down experiment. Upper panel: In vitro synthesized SAP97DPDZ1-2 and SAP97DPDZ1-3 (lanes 1 and 2, respectively) were affinity purified with glutathione-agarose bound GST-TACE (lanes 3 and 4, respectively) or with glutathione-agarose bound GST (lanes 5 and 6, respectively). Lower panel: Coomassie staining of GST-TACE cytoplasmic tail (lanes 3 and 4, position indicated by the arrow) and GST (lanes 5 and 6, position indicated by the arrow head).

View larger version (36K): [in a new window]   Fig. 2. The C-terminal extremity of TACE is involved in the binding with SAP97. (A) Interaction analyzed by yeast two-hybrid system. Full-length SAP97 and mutant forms of TACE were expressed as fusion proteins with a GAL4 activation domain and a GAL4 DNA-binding domain, respectively (see Materials and Methods) and are schematically represented in the lower part of the figure. (B) Western blot showing that TACE mutant forms fused to GAL4 DNA-binding domain (GAL4BD) are expressed in yeast. Detection was made using anti-GAL4BD monoclonal antibody.

View larger version (32K): [in a new window]   Fig. 3. Biochemical evidence that TACE and SAP97 interact in mammalian cells. (A,B) Interaction of endogenous TACE and SAP97. COS-7 cells lysate (lanes 1) was submitted to a mock immunoprecipitation (lanes 2) or to an immunoprecipitation using an anti-SAP97 antibody (lanes 3) as described in Materials and Methods. (A) Results were analyzed by immunoblotting under reducing conditions using the C-terminus-specific TACE antibody. The position of the immature (around 120 kDa) and mature (around 90 kDa) form of TACE is indicated by i and m, respectively. (B) Results were analyzed by immunoblotting under non-reducing conditions using the ectodomain-specific TACE antibody. The position of the mature form of TACE (around 80 kDa) is indicated by m; the arrow head indicates the position of the IgG. (C) PDZ3 domain of SAP97 interacts with immature and mature forms of TACE. Left: Lysates from cells transfected with GFP-SAP97 (lane 1) or GFP-SAP97DPDZ3 (lane 2) were submitted to immunoprecipitation using a anti-GFP antibody as described in Materials and Methods (lanes 3 and 4, respectively). Results were analyzed by immunoblotting using the C-terminal-specific TACE antibody. Right: Western blot on COS-7 cells lysates using anti-SAP97 antibodies attests for the expression of SAP97 and SAP97DPDZ3. (D) The PDZ-binding motif of TACE interacts with SAP97. Left: lysates from mock-transfected COS-7 cells (lane 1). Lysates from cells co-transfected with GFP-SAP97 and haTACE (lane 2) or with GFP-SAP97 and haTACEm (lane 3) were submitted to immunoprecipitation using anti-HA antibody as described in Materials and Methods (lanes 4 and 5, respectively). Results were analyzed by immunoblotting using an anti-SAP97 antibody. Right: Western blot of COS-7 cells lysates using anti-HA epitope antibody demonstrates the expression of haTACE and haTACEm. Overexpressed forms of TACE are schematically detailed: black (HA), hemagglutinin epitope; white, disintegrin-like domain (beginning at amino acid 475); gray (mb), transmembrane region; striped, cytoplasmic domain with mutations indicated.

View larger version (71K): [in a new window]   Fig. 4. Coexpression of TACE and SAP97 alters their cellular distribution. COS-7 cells were transfected with different forms of haTACE and GFP-SAP97 and their cellular distribution was analyzed. (A) GFP-SAP97 presents a distribution similar to that of GFP-SAP97DPDZ3. (B) haTACE presents a distribution similar to that of haTACEm. (C) Distribution of GFP-SAP97 when coexpressed with haTACE (three representative cellular distributions are shown). (D) Distribution of GFP-SAP97 when coexpressed with haTACEm. (E) Distribution of GFP-SAP97DPDZ3 when coexpressed with haTACE. Distribution of GFP-SAP97 overexpressed forms were assessed by GFP epifluorescence with a FITC filter. TACE overexpressed forms were detected using anti-HA epitope antibodies followed by Rhodamine-conjugated secondary antibodies and their distributions revealed by Rhodamine epifluorescence.

View larger version (45K): [in a new window]   Fig. 5. Overexpressed TACE and SAP97 are colocalized. COS-7 cells were transfected with haTACE and GFP-SAP97. Two examples of colocalization are shown. TACE: localization of the overexpressed haTACE. SAP97: localization of the overexpressed GFP-SAP97. TACE+SAP97: superimposition of the two previous images. Areas where TACE and SAP97 colocalized appeared in a deep yellow color.

View larger version (31K): [in a new window]   Fig. 6. Cellular distribution of overexpressed TACE. Lysates of COS-7 cells overexpressing haTACE were treated in vitro with PNGase F or Endo H as described in Materials and Methods and submitted to western blot analysis using the anti-HA epitope antibody to detect overexpressed haTACE (A) or the cytoplasmic-domain-specific TACE antibody to detect endogenous TACE (B). Mock (lanes 1 and 3) samples without PNGase F or EndoH. Samples treated with PNGase F (lanes 2) or with EndoH (lanes 4). Immature and mature forms of endogenous TACE are indicated by i and m, respectively. Overexpression of haTACE was analyzed by flow cytometry at the cell surface (C) or intracellularly (D) as described in Materials and Methods. Solid lines (unshaded areas) depict untransfected cells, and shaded areas haTACE transfected cells.

View larger version (90K): [in a new window]   Fig. 7. Endogenous TACE and SAP97 can colocalize. (A) Immunocytochemistry for the detection of endogenous TACE and SAP97 was performed, as described in Materials and Methods, in confluent COS-7 cells and CACO-2 cells. The localization was analyzed by immunofluorescence confocal microscopy using separate channels. TACE: localization of endogenous TACE. SAP97: localization of endogenous SAP97. TACE+SAP97: superimposition of the two previous images. Areas where TACE and SAP97 colocalized appeared in a deep yellow color. (B) Western blots of CACO-2 cells lysate. Lane 1 detection of TACE; lane 2 detection of SAP97. Sizes in kDa are indicated on the left.