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First published online December 1, 2003
doi: 10.1242/10.1242/jcs.00848

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The S. cerevisiae HtrA-like protein Nma111p is a nuclear serine protease that mediates yeast apoptosis

M. E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, 4056 Basel, Switzerland

View larger version (70K): [in a new window]   Fig. 1. Nma111p is a nuclear protein that interacts with the NPC. (A) Haploid cells whose endogenous Nma111p was C-terminally tagged with GFP were examined by direct immunofluorescence. A confocal fluorescence micrograph (left) and a coincident fluorescence/differential-interference-contrast image (right) is shown. (B) Immunogold-EM localisation of Nma111p-GFP at the NPC. Triton-X-100-extracted spheroplasts were preimmunolabelled with a polyclonal anti-GFP antibody directly conjugated to 8-nm colloidal gold. Selected examples of gold-labelled NPCs are shown in cross-sections along the NE. The antibody labelled the cytoplasmic face of the NPC (top) and the nuclear face of the NPC (bottom). c, cytoplasm; n, nucleus. (C) Quantitative analysis of the gold-particle distribution associated with the NPC relative to its two symmetry axes in Nma111p-GFP cells. The central plane of the NPC is within the NE, the eightfold symmetry axis is perpendicular to the plane of the NE. 68 gold particles were scored. Scale bars, 5 µm (A); 100 nm (B).

View larger version (40K): [in a new window]   Fig. 2. Deletion of nma111 increases the survival rate of yeast cells after shifting to elevated temperatures. The thermotolerance of wild-type and nma111 cells initially grown at 25°C was tested at 42°C and 50°C, respectively, for different durations. At 50°C, thermotolerance was also analysed by incubating the cells at 37°C for 30 minutes to induce the expression of heat-shock proteins before shifting the cells to 50°C. A serial dilution of cells was plated on YPAD plates and incubated for 2 days at 25°C. Plates show the number of surviving cells after heat treatment for 30 minutes, 60 minutes and 180 minutes.

View larger version (54K): [in a new window]   Fig. 3. nma111 null cells do not undergo apoptosis. Wild-type (BMA41a), nma111 mutant and yca1 control cells were treated with 3 mM H2O2 for 3 hours to induce apoptosis. DNA was visualised by Sytox-Green nucleic-acid stain. Wild-type, nma111 mutant and yca1 control cells were analysed for DNA strand breaks by the TUNEL test and ROS by DHR staining after treatment with 0.8 mM H2O2 for 4 hours. Confocal fluorescence micrographs and coincident fluorescence/differential-interference-contrast images are shown.

View larger version (184K): [in a new window]   Fig. 4. Electron micrographs of wild-type and mutant nma111 cells after induction of apoptosis with 3 mM H2O2. (A-D) Wild-type cells treated with H2O2 exhibit a more prominent endoplasmic reticulum (white arrowhead in A), chromatin condensation along the NE (black arrows in B,C), blebs from the NE (white arrow in C), and tiny vesicles on the outer face of the plasma membrane (black arrowheads in D). (E,F) nma111 cells do not exhibit any of these apoptotic hallmarks at the EM level. Scale bars, 500 nm.

View larger version (54K): [in a new window]   Fig. 5. Overexpression of ProtA-Nma111p leads to apoptotic-like cell death. (A) Extracts of cells carrying plasmid-borne N-terminally protein-A-tagged Nma111p that were grown in glucose medium for 4 hours or induced with galactose medium for 26 hours were immunoblotted and probed with an anti-protein-A antibody. (B) Survival rates of yeast cells overexpressing ProtA-Nma111 compared with nma111 cells without pretreatment or with incubation in 0.4 mM H2O2 for 24 hours. Data represent mean±s.d. (C) Sytox-Green DNA stain and TUNEL assay of ProtA-Nma111p overproducer. Scale bars, 5 µm (Sytox), 10 µm (TUNEL).

View larger version (106K): [in a new window]   Fig. 6. Nma111p-GFP aggregates inside the nucleus at elevated temperatures and after induction of apoptosis. In yeast cells grown at 25°C, Nma111p-GFP is evenly distributed throughout the nucleus (A), whereas, in cells shifted to 42°C (B) or 50°C (C), the protein forms aggregates inside the nucleus. Moreover, in cells shifted to 50°C a partial translocation of Nma111p-GFP into the cytoplasm was observed (D and E). In apoptotic cells (F), after treating cells with 3 mM H2O2 for 3 hours, Nma111p-GFP also aggregates inside the nucleus. Nma111p-GFP was visualised by direct immunofluorescence of the GFP tag. Under similar conditions, the localisation of the control protein Xpo1-GFP remains unaffected: (G) 25°C; (H) 42°C; (I) 50°C; (J) 3 mM H2O2 for 3 hours. Confocal fluorescence micrographs (left) and coincident fluorescence/differential-interference-contrast images are shown (right). Scale bars, 5 µm.

View larger version (61K): [in a new window]   Fig. 7. The apoptosis-mediating effect of Nma111p depends on its serine-protease activity. Indirect immunofluorescence localisation of ProtA-Nma111p (A) and ProtA-Nma111p-S235C (B) in yeast cells grown to mid-log phase. Sytox-Green nucleic-acid stain of ProtA-Nma111p (C), ProtA-Nma111p-S235C cells (D) and nma111 cells (E), as well as visualisation of DNA-strand breaks by TUNEL staining in ProtA-Nma111p (F), ProtA-Nma111p-S235C (G) and nma111 cells (H). Confocal fluorescence micrographs and coincident fluorescence/differential-interference contrast-images are shown. Scale bars, 5 µm (A-E), 10 µm (F-H). (I) Survival of ProtA-Nma111p, ProtA-Nma111p-S235C and nma111 cells grown to mid-log phase after incubation with H2O2 for 4 hours at indicated concentrations.