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Fig. 5. Identification of the giantin cleavage sites. (A) Schematic representation of the structure of giantin showing the putative caspase-3 cleavage sites that were mutated. The transmembrane domain is shown in black. Potential coiled coil-forming regions are shaded grey. (B) In vitro translated ³⁵S-labelled wild-type giantin or giantin with the indicated aspartic acid to alanine substitutions were incubated with control or apoptotic HeLa cytosol in the absence or presence of 2 µM Ac-DEVD-CHO for 4 hours at 37°C and analysed by SDS-PAGE and autoradiography. Full-length (FL) and defined caspase cleavage products (^*) are marked. Additional cleavage products (filled circles) are also shown. The arrowhead marks an additional faint band seen only with the D1882A mutant. (C) HeLa cells expressing GFP-tagged NAGT1 (NAGFP) and FLAG-tagged wild-type giantin, giantin D1083A, giantin D1882A, or the double point mutant were induced with 2 µM staurosporine for 8 hours to undergo apoptosis and visualized for NAGFP and FLAG staining by epifluorescence microscopy. Apoptotic cells are indicated with arrowheads. Scale bar, 10 µm. (D) Scheme to describe the likely order of cleavages occurring within giantin. The P1 and 2 products are produced first by cleavage at D¹⁸⁸². The P3 product and an additional unidentified fragment are generated by secondary cleavage of P1 at DAGD¹⁰⁸³.

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