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First published online 19 February 2003
doi: 10.1242/jcs.00290

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Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions

Department of Biochemistry and Biophysics, University of California San Francisco, Genentech Hall, 600 16th Street, San Francisco, California, 94143-2140, USA

View larger version (24K): [in a new window]   Fig. 7. The C2B of synaptotagmin VII contains a dominant and transplantable inhibitory subdomain. Chimeric constructs were generated that replaced ß-strands of the eight-stranded ß-barrel in pairs. The resulting chimeras fell into two categories: a synaptotagmin VII C2B with (A) increasing numbers of N-terminal synaptotagmin-I-derived ß-strands [7B series (F7B-H7B)] and (B) the inverse series [1B series (F1B-H1B)]. The construct compositions are as follows: F7B (synaptotagmin I residues 266-303 fused to synaptotagmin VII residues 298-403), G7B (synaptotagmin I residues 266-334 fused to synaptotagmin VII residues 329-403), H7B (synaptotagmin I residues 266-369 fused to synaptotagmin VII residues 364-403), F1B (synaptotagmin VII residues 261-297 fused to synaptotagmin I residues 304-421), G1B (synaptotagmin VII residues 261-328 fused to synaptotagmin I residues 335-421), H1B (synaptotagmin VII residues 261-363 fused to synaptotagmin I residues 370-421). PC12 cells expressing these chimeras were subsequently studied for their capacity to endocytose the proteins with the endocytosis assay. (C) A model of the synaptotagmin VII C2B domain depicts the relative positions of the inhibitory region (red), the AP-2-binding site (cyan) and the WHXL motif (green).

View larger version (39K): [in a new window]   Fig. 1. Synaptotagmin VII is not endocytosed in PC12 cells or CHO cells. The CD4-C2A-C2B constructs of synaptotagmin I (Syt 1) and synaptotagmin VII (Syt 7) were tested for their ability to be endocytosed. The cytoplasmic domains corresponded to residues 95-421 of full-length synaptotagmin I and residues 98-403 of full-length synaptotagmin VII. (A) The internalization was tested by surface labeling the cells for 1 hour at 4°C with 125I-Q4120, directed against the CD4 epitope, and next incubating at 37°C for 10 minutes to allow endocytosis before returning the cells to 4°C. Internalized 125I-Q4120 was determined by acid stripping the remaining surface label and lysing cells to quantify internalized counts. The fraction internalized was calculated by dividing the internal counts by the total cell associated counts (surface counts plus internalized counts). A background of cells kept at 4°C was subtracted from this value. This fraction internalized was then converted to an internalization index by subtracting the non-specific internalization of the CD4-Tailless construct and by normalizing to the extent of internalization of the Syt 1 construct in PC12 cells. (B) CHO cells stably expressing the CD4-C2A-C2B constructs were also analyzed using this internalization assay; the internalization is compared in terms of fraction internalized relative to the CD4-Tailless construct. (C) The C2A-C2B domains of synaptotagmins I and VII are aligned to show conserved sequence elements including the AP-2-binding site and the C-terminal WHXL. The asterisks (*) indicate Ca2+-binding residues known to influence dimerization and internalization of synaptotagmin I, and the residue numbers correspond to the position of the amino acids within the full-length protein.

View larger version (15K): [in a new window]   Fig. 2. The CT of synaptotagmin VII can be internalized in a tryptophan-dependent manner. (A) The CD4-CT constructs of synaptotagmin I (residues 393-421, CT 1) and synaptotagmin VII (387-403, CT 7) were tested for their ability to be endocytosed in PC12 cells using the previously described internalization assay. (B) When the tryptophan residue with the C-terminal WHXL is mutated to an alanine in either CT1 (W404A) or CT 7 (W398A), the internalization in PC12 cells is abolished.

View larger version (13K): [in a new window]   Fig. 3. The C2A of synaptotagmin VII is internalized in a cell-type-independent and tryptophan-independent manner. Using the previously described internalization assay, the CD4-C2A constructs of synaptotagmin I (residues 95-265, C2A 1) and synaptotagmin VII (residues 98-260, C2A 7) were tested. PC12 cells (A) and CHO cells (B) stably expressing the constructs were both analyzed by this method. (C) A point mutant changing the tryptophan residue of C2A 7's WKXL (W253A) to an alanine did not affect the strong endocytosis of C2A 7 in PC12 cells.

View larger version (45K): [in a new window]   Fig. 4. The CT and C2A of synaptotagmin VII are taken up into an internal compartment in PC12 cells. A morphological uptake assay was used to verify the results seen with the radioactive internalization assays. In this case, PC12 cells stably expressing Syt 7 (A,B), CT 7 (C,D) and C2A 7 (E,F) were plated on eight-well slides, surface stained with anti-CD4 antibody at 4°C (A,C,E) and moved to 37°C for 10 minutes (B,D,F). Cells were fixed, and immunofluorescence microscopy was carried out.

View larger version (25K): [in a new window]   Fig. 5. The CT and C2A of synaptotagmin VII are internalized by different pathways in PC12 cells. PC12 cells stably expressing either CT 7 (A,B) or C2A 7 (C,D) were transiently transfected with wild-type dynamin or dominant-negative dynamin K44E (A,C) or with control eps15 epsD32 or dominant-negative eps15 eps95/295 (B,D). The x-axis of the histograms shows the level of cell surface staining in each case. The curve for the wildtypes or controls are shown in black, and the curves for the dominant-negative mutants are shown in red. The mean of this value was taken and used to generate the calculated factor `cell surface fold increase' by dividing the mean surface staining in the dominant negative by the mean of the wildtype or control case (E).

View larger version (13K): [in a new window]   Fig. 6. The C2B of synaptotagmin VII is not internalized in PC12 cells. PC12 cells were generated that stably expressed CD4-C2B constructs for synaptotagmin I (residues 266-421, C2B 1) and synaptotagmin VII (residues 261-403, C2B 7). These cells were examined by the previously described radioactive internalization assay.