Expression of Syn7. (A) Total membrane fractions prepared from rat brain (B), liver (L), kidney (K), spleen (S), testis (T), and MDCK (M) cells. Aliquots (10 μg of protein) were subjected to SDS-PAGE and immunoblotting with affinity-purified rabbit anti-Syn7 polyclonal antibody (rabbit anti-Syn7#1). Total membrane fraction from MDCK cells (10 μg of protein) was immunoblotted with an affinity-purified goat anti-Syn7 polyclonal antibody (B) or a separate rabbit antibody to Syn7 (rabbit anti-Syn7#2) (C). (D) Total membrane fraction (10 μg protein/lane) from a clone of NRK cells (NRK-K2) stably transfected with HA epitope–tagged Syn7 was subjected to immunoblot analysis with monoclonal anti-HA antibodies or rabbit anti-Syn7#1. (E–J) Rabbit anti-Syn7#1 (E–G) or goat anti-Syn7 antibodies (H–J) were incubated with no additions (E and H), 300 μg/ml GST–Syn13 (F and I), or 300 μg/ml GST–Syn7 (G and J) before labeling MDCK cells. Images were collected from a single focal plane and normalized to control. (K–M) Subconfluent MDCK cells were labeled for Syn7 with Alexa 488–conjugated secondary antibodies (top) in combination with antibodies specific for TfR (K), EEA1 (L), or CI-MPR (M) with Texas Red–conjugated secondary antibodies (bottom). Syn7 was labeled with the rabbit anti-Syn7#1 (K or L) or with the goat anti-Syn7 antibody (M). Cells were imaged by collecting a 4-μm-thick z-series (step, 0.1 μm) and subjecting the series to computed deconvolution before assembling a maximum point projection representing a 1-μm-thick plane. Arrows point to structures that are positive for two markers, and arrowheads point to structures that are positive for only one marker. N, nucleus. Bars, 15 μm for E–J; 5 μm for K–M.

Localization of epitope-tagged Syn7 in NRK cells by confocal microscopy. Stably transfected NRK cells expressing an HA epitope–tagged Syn7 (NRK-K2 cells) were labeled for HA with the use of monoclonal anti-HA antibodies (A, C, and D, left) or rabbit anti-HA polyclonal antibodies (B, left) in combination with rabbit polyclonal antibodies to Syn13 (A, middle), mouse mAbs to TfR (B, middle), rabbit polyclonal antibodies to lgp120 (C, middle), or rabbit polyclonal antibodies to EEA1 (D, middle). E shows labeling with anti-lgp120 rabbit polyclonal antibodies (left) with anti-EEA1 mAbs (middle). Images were collected from a 0.5-μm focal plane by confocal microscopy. Note that variations in the distribution of Syn7 result from the different focal planes selected to show the localization of epitope-tagged Syn7 relative to each marker protein. Green/red merged images are shown on right. Bar, 10 μm.

Immunogold electron microscopy of Syn7 and Vamp 8 associated with rat liver late endosomes, lysosomes, and hybrid organelles. Immunoelectron microscopy of Syn7 (A–C) labeling with the use of rabbit anti-Syn7#1 (15-nm gold; large arrowheads), Vamp 8 (E–G) labeling (15-nm gold; large arrowheads), and internalized ASF-avidin (A, B, E, and G) (8-nm gold; small arrowheads) associated with rat liver late endosomes (A and E), hybrid organelles (B and F), and lysosomal fractions (C and G). Late endosomes and hybrid organelles were identified by their content of ASF–avidin. Lysosomes were identified by their electron-dense morphology. Bar, 200 nm. (D and H) Quantitation of the level of Syn7 labeling (D) and Vamp 8 labeling (H) on the peripheral membrane of late endosomes (le; 30 μm scored), hybrid organelles isolated after late endosome–lysosome fusion in the cell-free fusion system (h; 65 μm scored), and lysosomes isolated after incubation with cytosol and ATP under the conditions of the cell-free fusion system (ly; 1000 μm scored). Error bars represent SEM for the number of organelles scored.

Immunofluorescence localization and coimmunoprecipitation of Vamp 8 with Syn7. (A) Specificity of Vamp 8 antibodies was demonstrated by subjecting whole cell extracts of MDCK cells to SDS-PAGE and immunoblotting with affinity-purified rabbit anti-Vamp 8 antibodies. (B) A Triton X-100–soluble fraction was prepared from a postnuclear supernatant from MDCK cells and allowed to incubate with the affinity-purified rabbit anti-Syn7 antibody or the rabbit anti-GST antibody (left panels) or the affinity-purified goat anti-Syn7 antibody and a matching goat anti-GST antibody (right panels). Fixed S. aureus cells were then added, and immunoprecipitates were washed, subjected to nonreduced SDS-PAGE, and immunoblotted for Syn7, Vamp 8, and Vamp 3. One-fifth of the corresponding supernatant from the immunoprecipitate was also immunoblotted. For immunoprecipitations with the rabbit anti-Syn7#1 antibody, the goat anti-Syn7 antibody was used for immunoblot analysis. For immunoprecipitations with the goat anti-Syn7 antibody, the rabbit anti-Syn7#1 antibody was used for immunoblot analysis. SDS-PAGE was performed under nonreducing conditions. (C) MDCK cells were labeled with goat anti-Syn7 (left) and rabbit anti-Vamp 8 (right) in combination with FITC-conjugated donkey anti-goat and Texas Red–conjugated donkey anti-rabbit secondary antibodies. Matched 1-μm-thick planes from a deconvoluted 4-μm-thick z-series are shown (D). Polarized WIF-B cells were fixed and permeabilized, double labeled with rabbit anti-Vamp 8 (left) and mouse anti-lgp120 (right), and imaged by confocal microscopy. Primary antibodies were followed by Cy3- and FITC-conjugated secondary antibodies, respectively. In C and D, arrows point to structures that are positive for both markers, and arrowheads indicate structures that contain only one marker. BC, bile canalicular-like space; N, nucleus. Bar, 10 μ m.

Distribution of Syn7 and Syn13 in WIF-B cells by confocal microscopy. Polarized WIF-B cells were fixed and permeabilized and then double-labeled with antibodies to Syn7 and lgp120 (A), Syn13 and lgp120 (B), or Syn13 and TfR (C). Mouse mAbs were used to label Syn7 (A), lgp120 (B), and TfR (C) with rabbit polyclonal antibodies labeling lgp120 (A) and Syn13 (B and C). FITC-conjugated antibodies to mouse IgG (A, Syn7; B, lgp120; C, TfR) and Cy3-conjugated antibodies to rabbit IgG (A, lgp120; B and C, Syn13) were used as secondary antibodies. Optical sections in A and B are through the middle of the cells, whereas the section in C is slightly closer to the substratum to show more peripheral endosomes. Arrows point to structures that are positive for two markers, and arrowheads point to structures that are positive for only one marker. Note the good coincidence of Syn7 with lgp120-positive structures, i.e., lysosomes (A), and of Syn13 with TfR, i.e., early endosomes (C), whereas little overlap is seen between Syn13 and lgp120 (B). BC, bile canalicular-like space; N, nucleus. Bar, 10 μm.

Immunogold electron microscopic localization of Syn7 in MDCK cells. (A) Ultrathin cryosections were labeled with a specific affinity-purified antibody against Syn7 (anti-Syn7#1) followed by 15-nm protein A–gold. Specific labeling (arrowheads) was observed in the perinuclear region of the cell associated with multivesicular endosomes (e). (B–D) Intracellular MDCK membranes were fixed with paraformaldehyde and adsorbed to Formvar-coated grids. Membranes were then sequentially double labeled for Syn7 (15-nm protein A–gold; S7) and CI-MPR (20-nm protein A–gold; MPR) (B); Syn7 (20-nm protein A–gold; S7) and Rab7 (10-nm protein A–gold; R7) (C); or Syn7 (10-nm protein A–gold; S7) and EEA1 (15-nm protein A–gold) (D). Bar, 300 nm

Syn7 antibodies specifically inhibit late endosome–lysosome fusion in vitro. (A) Titration experiment assaying content mixing between rat liver late endosomes and lysosomes. Effects of affinity-purified rabbit antibodies to Syn7#1 (closed squares) and Syn7#2 (closed circles) and rabbit antibodies to the cytosolic tail of the CI-MPR (open circles) were measured after treating the two fractions separately with the indicated concentrations of antibodies for 15 min on ice and keeping the antibodies present during the cell-free late endosome–lysosome fusion assay. Standard fusion (100%) is defined as the amount of fusion obtained after preincubation with buffer only, i.e., without the addition of antibodies. Data are presented as means ± SEM of duplicate samples from six separate experiments for anti-CI-MPR, five separate experiments for anti-Syn7#1 at 20 μg/ml, four separate experiments for anti-Syn7#1 at 40 μg/ml, three separate experiments for anti-Syn7#2 at 20 μg/ml, and two separate experiments for anti-Syn7#1 at 80 μg/ml. All other data presented are means of duplicate samples. (B) Summation of several experiments with the indicated antibodies to perturb the fusion assay between late endosomes and lysosomes. Before fusion, both late endosomes and lysosomes were preincubated with buffer only (Control) or the following antibodies at 40 μg/ml: anti-CI-MPR, affinity-purified antibodies to the cytosolic tail of the CI-MPR; anti-Syn7#1, affinity-purified antibodies to the cytosolic domain of Syn7 from serum #1; absbd anti-Syn7#1, preincubation with anti-Syn7#1 previously absorbed with GST–Syn7 (equivalent to anti-Syn7#1 in the assay at 40 μg/ml); anti-Syn7#2, affinity-purified antibodies to the cytosolic domain of Syn7 from serum #2; anti-syntaxin 6, affinity-purified antibodies to the cytosolic domain of syntaxin 6; and anti-GST, affinity-purified antibodies (40 μg/ml) to GST. Anti-Syn7(Fab), Fab fragments prepared from anti-Syn7#2 antibody were used at 45 μg/ml. Data presented are means of duplicate samples or means ± SEM in cases of three or more separate experiments.