Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.
Animals ; Apoptosis ; Cell Line, Tumor ; Cell Membrane Permeability ; Humans ; Membrane Potential, Mitochondrial ; physiology ; Mice ; Mitochondrial Membrane Transport Proteins ; physiology ; Mitochondrial Membranes ; metabolism ; Mutation ; Necrosis ; Proteins ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Rhodamine 123 ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; physiology ; bcl-X Protein ; metabolism