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.

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