Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Bruno Antonsson,Gorka Basañez,Olatz Landeta,Oihana Terrones,Ane Landajuela,Aitor Etxebarria,Hong-Gang Wang,Hirohito Yamaguchi

doi:10.1074/jbc.m808050200

Abstract

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

Highlights

Multiple proteins implicated in mitochondrial morphogenesis during normal growth conditions can cross-talk with BCL-2 family members to affect the mitochondrial pathway of apoptosis [4]
We switched to a quantitative liposome release assay that faithfully reproduces basic aspects of the BAX-driven MOM permeabilization (MOMP) pathway in which mitochondrial outer membrane (MOM)-like large unilamellar vesicles (LUV) are preloaded with FD70
The Ability of BAX-interacting factor 1 (Bif-1) N-BAR to Produce Large Scale Morphological Rearrangements in MOM-like LUV Can Be Uncoupled from Its Stimulatory Effect on BAX-permeabilizing Function—Because interactions between N-BAR-containing proteins and anionic liposomes can produce global changes in vesicle morphology (14 –17), we examined whether Bif-1 possesses the ability to modify the morphology of MOM-like LUV and whether this property is mechanistically linked to potentiation of BAX-permeabilizing function

Summary

EXPERIMENTAL PROCEDURES

Materials—Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), liver phosphatidylinositol (PI), heart cardiolipin (CL), myristoylated cardiolipin (myrCL), monolysocardiolipin (MCL), dilysocardiolipin (DCL), N-(NBD)-phosphatidylethanolamine), and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine were purchased from Avanti Polar Lipids (Alabaster, AL). Proteins were incubated with LUV (50 ␮M) for 10 min in a thermostatically controlled 1-cm path length cuvette with constant stirring at 37 °C (Vfinal ϭ 750 ␮l) followed by determination of extents of intervesicular lipid mixing. Immunoprecipitations—Proteins were incubated with LUV (100 ␮M) in KHE buffer for 30 min at 37 °C under constant stirring using an Eppendorf Thermomixer (Vfinal ϭ 300 ␮l) followed by the addition of CHAPS (0.5% w/v, final concentration) and immunoprecipitation with agarose-conjugated anti-BAX N20 antibody (Santa Cruz) for 2 h at 4 °C on a rotator. LUV (50 ␮M) were incubated with or without proteins in KHE buffer for 10 min at 37 °C under constant stirring using an Eppendorf Thermomixer (Vfinal ϭ 70 ␮l) followed by DLS analysis. Data were analyzed by the cumulant method using the software provided by the instrument

RESULTS

Liposome sizeb

DISCUSSION