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First published online 11 April 2006
doi: 10.1242/jcs.02902

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The inhibitor-of-apoptosis protein Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast homologue of Omi/HtrA2

David Walter^1,*, Silke Wissing^2,*, Frank Madeo^2,3 and Birthe Fahrenkrog^1,

¹ M. E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
² Institute for Physiological Chemistry, University of Tübingen, 72076 Tübingen, Germany
³ IMB, Karl-Franzens University, Universitätsplatz 2, 8010 Graz, Austria

View larger version (29K): [in a new window]   Fig. 1. (A) Western blot analysis of Bir1p-GFP expression. Bir1p-GFP cells transformed with either plasmid-borne N-terminally protein-A-tagged Nma111p or Nma111-S235C were grown in glucose medium and subsequently shifted to galactose medium for 24 hours. Bir1p-GFP cells grown in YPAD were used as control cells. Cell lysates were prepared, immunoblotted and probed with a monoclonal anti-GFP antibody. (B) In vitro binding-study of Nma111p and Bir1p. Recombinantly expressed GST-Bir1p and GST were incubated with in vitro-synthesized 35S-labelled Nma111p-fragments. Unbound and bound fractions were analysed by SDS-PAGE and autoradiography. (C) In vitro binding study of Yca1p, Nma111p and Bir1p. Recombinantly expressed GST-Bir1p, GST-Nma111p and GST were incubated with in vitro-synthesized 35S-labelled Yca1p. Unbound and bound fractions were analysed by SDS-PAGE and autoradiography.

View larger version (68K): [in a new window]   Fig. 2. Bir1p localizes to the cytoplasm and the nucleus of yeast cells. Confocal fluorescence micrographs (top), and coincident fluorescence and differential-interference contrast images (bottom) are shown. Haploid cells, whose endogenous Bir1p was C-terminally tagged with GFP and transformed with pYES-ProtA-Nma111p, were examined by direct fluorescence after induction of Nma111p overexpression (left panels). bir1 cells were transformed with pNOPPATA1W-Bir1p to express N-terminal protein-A-tagged Bir1p (ProtA-Bir1p) and examined by indirect immunofluorescence using a primary polyclonal anti-protein-A antibody and a secondary anti-rabbit IgG antibody labelled with Alexa Fluor 488 (middle panels). Haploid cells that were genomically tagged with a tandem-affinity purification tag at the C-terminus of endogenous Bir1p (Bir1p-TAP) and analysed by indirect immunofluorescence using a primary anti-protein A antibody and a secondary antibody labelled with Alexa Fluor 488 (right panels). Bars, 5 µm.

View larger version (61K): [in a new window]   Fig. 3. bir1 cells undergo apoptosis. Confocal fluorescence (first and third column of panels), and coincident fluorescence and differential-interference-contrast (second and fourth column of panels) images are shown. (A) Wild-type (BYa) and bir1 cells were grown in YPAD medium, treated with 0.8 mM H2O2 for 4 hours and analysed for apoptotic hallmarks. DNA was visualized by Sytox-Green nucleic acid stain. DNA-strand breaks were detected by the TUNEL test and ROS by DHR staining. Bars, 2.5 µm (DNA); 5 µm (ROS); 10 µm (TUNEL). (B) Survival of wild-type (BYa) and bir1 cells after incubation with H2O2 for 4 hours at indicated concentrations. Error bars, mean + s.e.m.

View larger version (53K): [in a new window]   Fig. 4. Overexpression of Bir1p prevents apoptosis-like cell death. Confocal fluorescence (first, third and fifth column of panels), and coincident fluorescence and differential-interference-contrast (second, fourth and sixth column of panels) images are shown. (A) Wild-type cells (BYa), cells overexpressing Bir1p and cells simultaneously overexpressing Bir1p and Nma111p were treated with 0.8 mM H2O2 for 4 hours to induce apoptosis. DNA was visualized by Sytox-Green nucleic acid stain. DNA-strand breaks were detected by the TUNEL test and ROS by DHR staining. Bars, 2.5 µm (DNA), 5 µm (ROS); 10 µm (TUNEL). (B) Survival rates of yeast cells overproducing Bir1p compared with overproducers of Bir1p/Nma111p or Nma111p, without pre-treatment or after incubation with 0.4 mM H2O2 for 24 hours. Error bars, mean + s.e.m.

View larger version (150K): [in a new window]   Fig. 5. Electron micrographs of Bir1p-overexpressing cells and bir1 cells. (A-B) Cells overexpressing Bir1p that had been treated with 0.8 mM H2O2 for 4 hours do not show any apoptotic hallmarks. (C-F) bir1 cells show strong invaginations of the nuclear envelope (black arrows in C), a very prominent endoplasmatic reticulum (white arrowheads in D and F), chromatin condensation at the nuclear envelope (black arrowheads in D), and tiny vesicles on the outer face of the plasma membrane (grey arrowheads in E). In few cells chromosome segregation defects were also detected (white arrows in F). Bars, 500 nm.

View larger version (45K): [in a new window]   Fig. 6. Chronological ageing of wild-type, bir1-, nma111- and Bir1p-overexpressing cells. (A) Survival rates. Error bars, mean + s.e.m. (B) TUNEL assay and ROS detection of wild-type (BYa), bir1-, nma111- and Bir1p-overexpressing cells after 5 days of cultivation. Bars, 5 µm.

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