Apoptosis

, Volume 15, Issue 5, pp 553–562

Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane

  • Vidyaramanan Ganesan
  • Meenu N. Perera
  • David Colombini
  • Debra Datskovskiy
  • Kirti Chadha
  • Marco Colombini
Original Paper

DOI: 10.1007/s10495-009-0449-0

Cite this article as:
Ganesan, V., Perera, M.N., Colombini, D. et al. Apoptosis (2010) 15: 553. doi:10.1007/s10495-009-0449-0

Abstract

A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.

Keywords

ApoptosisMitochondriaCeramideChannelBcl-2Protein release

Supplementary material

10495_2009_449_MOESM1_ESM.tif (19.2 mb)
Fig. S1: LaCl3 was added to an activated Bax-enhanced ceramide channel (2 μg ceramide and 11 nM activated Bax). Disassembly occurred after 1.8 min. The experiment shown is representative of more than 8 experiments. Amounts of added ceramide and ac-Bax were different between experiments. (TIFF 19,699 kb)
10495_2009_449_MOESM2_ESM.tif (12.5 mb)
Fig. S2: SDS–PAGE showing purity of recombinant Bax preparation. (TIFF 12,806 kb)
10495_2009_449_MOESM3_ESM.tif (3.5 mb)
Fig. S3: SDS–PAGE showing restricted digestion of activated Bax by trypsin. The digested fragment is 15 kDa as expected for detergent activated Bax. (TIFF 3,577 kb)

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Vidyaramanan Ganesan
    • 1
  • Meenu N. Perera
    • 2
  • David Colombini
    • 2
  • Debra Datskovskiy
    • 2
  • Kirti Chadha
    • 2
  • Marco Colombini
    • 1
    • 2
  1. 1.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA
  2. 2.Department of BiologyUniversity of MarylandCollege ParkUSA