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First published online 17 February 2004
doi: 10.1242/jcs.00950

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Caspase-mediated cleavage of syntaxin 5 and giantin accompanies inhibition of secretory traffic during apoptosis

Martin Lowe, Jon D. Lane^*, Philip G. Woodman and Victoria J. Allan

School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (57K): [in a new window]   Fig. 1. A subset of Golgi-associated transport factors are cleaved in apoptotic extracts. (A) Isolated rat liver Golgi membranes were incubated at 37°C with apoptotic HeLa cytosol in the absence or presence of 2 µM Ac-DEVD-CHO (D) for the times indicated. Membranes were pelleted and membrane and supernatant (cytosol) fractions analysed by immunoblotting with antibodies to giantin, GM130, syntaxin 5, GS28, or mannosidase I (Mann-1). Full-length proteins (FL) and caspase cleavage products (*) are marked. The major giantin cleavage products are denoted P1-P3. Minor giantin cleavage products are indicated with filled circles. (B) Golgi membranes were incubated at 37°C for 4 hours with control HeLa cytosol, apoptotic cytosol, or apoptotic cytosol in the presence of 2 µM Ac-DEVD-CHO. Samples were fractionated as in A and analysed by immunoblotting with antibodies raised against fragments encompassing the entire protein (mix) or the N terminus (NT) of giantin. The x indicates an unknown cytosolic protein that cross-reacts with the N-terminal antibody. (C) Golgi membranes were incubated at 37°C for 6 hours with control HeLa cytosol, apoptotic cytosol, or apoptotic cytosol in the presence of 2 µM Ac-DEVD-CHO. Samples were fractionated as in A and analysed by immunoblotting for a number of Golgi-associated proteins, as indicated. Blotting with three different polyclonal antibodies to p115 gave identical results.

View larger version (45K): [in a new window]   Fig. 2. Giantin and syntaxin 5 are rapidly cleaved during apoptosis in vivo. (A) HeLa cells were treated with 1 µM staurosporine in the absence or presence of 50 µM zVAD.fmk (Z) for the times indicated and analysed by immunoblotting with antibodies to PARP, giantin, GM130, syntaxin 5 and GS28. (B) HeLa cells were treated with anisomycin for 12 hours. Apoptotic cells became detached from the culture dish and these floating cells (F) were harvested by centrifugation. Remaining adherent cells (A) were harvested in parallel, and samples from each population were analysed by immunoblotting with antibodies to PARP, giantin, GM130, syntaxin 5 and GS28. Full-length (FL) and caspase cleavage products (*) are marked. The major giantin cleavage products P1-P3 are shown. Minor cleavage products are indicated with filled circles. (C) Confocal images of HeLa cells treated with UV radiation to induce apoptosis. Cells were incubated for 4 hours post-irradiation and stained with DAPI (blue), antibodies to GM130 (green) and antibodies raised against either the N terminus of giantin (NT) or fragments encompassing the entire protein (mix; red). Regions of overlap between GM130 and giantin are in yellow. Apoptotic cells are marked with arrowheads. Scale bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 3. Giantin and syntaxin 5 are cleaved by caspase-3. (A) Cytosols from HeLa cells were pre-incubated in the absence (control) or presence (apoptotic) of 10 µM cytochrome c for 90 minutes at 37°C to activate endogenous caspases. Golgi membranes were incubated with these control or apoptotic cytosols in the absence or presence of 80 µg/ml CasputinTM for 4 hours at 37°C and analysed by SDS-PAGE and immunoblotting. Full-length (FL) and caspase cleavage products (*) are marked. Filled circles indicate minor giantin cleavage products. (B) In vitro translated 35S-labelled control proteins, giantin or syntaxin 5 were incubated with purified recombinant caspases at the indicated concentrations for 2 hours at 30°C prior to SDS-PAGE and autoradiography. Defined caspase cleavage products (*) are marked. Additional cleavage products that may be non-specific (filled circles) are also marked. The open triangle indicates an additional giantin cleavage product seen only with caspase-2 that may be the cleavage partner for P3 (see also Fig. 5C). Control proteins were PARP (for caspase-3 and -7 at 0, 0.025, 0.1, 0.25, 0.5, 1, 2 and 4 nM) and pro-caspase-2 (for caspase-2 at 0, 0.5, 1, 2.5, 10, 25, 100, 250 nM).

View larger version (36K): [in a new window]   Fig. 4. Identification of the syntaxin 5 cleavage sites. (A) Schematic representation of the structure of syntaxin 5 isoforms showing the putative caspase-3 cleavage sites. Transmembrane domains are indicated in black and coiled coil-forming regions are shaded grey. The actual cleavage site is underlined. (B) In vitro translated 35S-labelled wild-type syntaxin 5 or syntaxin 5 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 caspase cleavage products (*) are marked. Note that the single point mutations give rise to a slight shift in mobility of the full-length protein.

View larger version (45K): [in a new window]   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.

View larger version (20K): [in a new window]   Fig. 6. The early secretory pathway is blocked in apoptotic cells. (A) (Top panel) HeLa cells were transfected with tsO45 VSV-G-GFP and incubated overnight at 39.5°C, then incubated in parallel for 2 hours at 39.5°C or 31°C as shown. They were stained without permeabilization, using anti-VSV-G, to score for transport to the cell surface. (Middle panel) Transfected cells were induced to enter apoptosis by UV irradiation and incubated for a further 4 hours at 39.5°C. Cells were either left at 39.5°C or shifted to 31°C for 2 hours, as indicated, then fixed in PFA. Apoptotic cells were identified using DAPI. (Bottom panel) As in middle panel, but with apoptosis induced by addition of 2 µM staurosporine. Results are presented as the mean ± s.e.m. (n=13-45). (B) Fluorescence microscopy of tsO45-VSV-G-GFP trafficking. UV-treated HeLa cells expressing VSV-G-GFP were treated as in A, and examined using confocal microscopy. The non-apoptotic cells show strong surface labelling (-VG), whilst the apoptotic cell (arrowhead) lacks surface label. Scale bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 7. VSV-G-GFP accumulates in the endoplasmic reticulum of apoptotic cells. (A) HeLa cells were transfected with tsO45 VSV-G-GFP, incubated overnight at 39.5°C and shifted to 31°C for 25 minutes. After fixation and permeabilisation, cells were stained with antibodies to the Golgi apparatus (GM130). Scale bar, 10 µm. (B) Transfected HeLa cells were incubated in the presence of 2 µM staurosporine for an additional 4 hours at 39.5°C to induce apoptosis prior to shifting to 31°C for 25 minutes. Cells were fixed, permeabilised and stained with the ER marker calnexin (red) and DAPI (blue). VSV-G-GFP is green and regions of overlap with calnexin are yellow. The images are three separate confocal slices taken through the middle region of the cell. Scale bar, 10 µm.