scholarly journals Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization

2008 ◽  
Vol 412 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Anthony J. Valentijn ◽  
John-Paul Upton ◽  
Andrew P. Gilmore

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

2011 ◽  
Vol 166 (1) ◽  
pp. 47-62 ◽  
Author(s):  
Jiri Dresler ◽  
Jana Klimentova ◽  
Jiri Stulik

2006 ◽  
Vol 1 (1) ◽  
pp. 418-428 ◽  
Author(s):  
Ilka Wittig ◽  
Hans-Peter Braun ◽  
Hermann Schägger

2017 ◽  
Vol 112 (3) ◽  
pp. 477a
Author(s):  
Anke Dopychai ◽  
Ralf Hausmann ◽  
Linda Krüger ◽  
Stefan Gründer ◽  
Günther Schmalzing

2004 ◽  
Vol 121 (4) ◽  
pp. 546-555 ◽  
Author(s):  
Jonas Kjell ◽  
Allan G. Rasmusson ◽  
Hakan Larsson ◽  
Susanne Widell

2008 ◽  
pp. 557-569
Author(s):  
Holger Eubel ◽  
A. Harvey Millar

PROTEOMICS ◽  
2012 ◽  
Vol 12 (17) ◽  
pp. 2729-2738 ◽  
Author(s):  
Adelson Joel da Silva ◽  
Diana Paola Gómez-Mendoza ◽  
Magno Junqueira ◽  
Gilberto Barbosa Domont ◽  
Edivaldo Ximenes Ferreira Filho ◽  
...  

2012 ◽  
Vol 445 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Yusuke Kakizaki ◽  
Anthony L. Moore ◽  
Kikukatsu Ito

Symplocarpus renifolius and Arum maculatum are known to produce significant heat during the course of their floral development, but they use different regulatory mechanisms, i.e. homoeothermic compared with transient thermogenesis. To further clarify the molecular basis of species-specific thermogenesis in plants, in the present study we have analysed the native structures and expression patterns of the mitochondrial respiratory components in S. renifolius and A. maculatum. Our comparative analysis using Blue native PAGE combined with nano LC (liquid chromatography)-MS/MS (tandem MS) has revealed that the constituents of the respiratory complexes in both plants were basically similar, but that several mitochondrial components appeared to be differently expressed in their thermogenic organs. Namely, complex II in S. renifolius was detected as a 340 kDa product, suggesting an oligomeric or supramolecular structure in vivo. Moreover, the expression of an external NAD(P)H dehydrogenase was found to be higher in A. maculatum than in S. renifolius, whereas an internal NAD(P)H dehydrogenase was expressed at a similar level in both species. Alternative oxidase was detected as smear-like signals that were elongated on the first dimension with a peak at around 200 kDa in both species. The significance and implication of these data are discussed in terms of thermoregulation in plants.


2002 ◽  
Vol 159 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Damien Arnoult ◽  
Philippe Parone ◽  
Jean-Claude Martinou ◽  
Bruno Antonsson ◽  
Jérôme Estaquier ◽  
...  

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.


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