Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants.
Bax, a proapoptotic member of the Bcl-2 family of proteins, resides in the cytosol and translocates to the mitochondrial membrane upon induction of apoptosis. It has been proposed that Bax does not translocate to mitochondria under normal physiological conditions, due to interaction between amino (ART) and carboxy (TM) terminal domains. Here, we report the physiological consequences of introducing a matrix targeting mitochondrial signal sequence (Su9) at the amino terminus of Bax and its mutants lacking ART, TM, or both segments. In vitro mitochondrial protein import assays of the fusion proteins suggests localization to the mitochondrial matrix. When expressed in Cos-1 cells, Su9 could target Bax to mitochondria in the absence of an apoptotic stimulus. However, mitochondrial localization did not result in apoptosis. When ART, TM, or both segments of Bax were deleted, expression of fusion proteins containing Su9 resulted in apoptosis via cytochrome c release. Cell death was inhibited by the pan-caspase inhibitor zVAD-fmk. We thus demonstrate that an effective mitochondrial matrix targeting signal can override the inhibition of import of Bax to the organelle, presumed to arise as a result of interaction between ART and TM segments, in the absence of apoptotic stimulus. We also demonstrate the ability of truncated variants of Bax to cause apoptosis when targeted to mitochondria by cytochrome c release from an ectopic environment.
John
George Babu
GB
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Anjum
Rana
R
Khar
Ashok
A
Nagaraj
Ramakrishnan
R
D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002451:Cell Compartmentation; D003574:Cytochrome c Group / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014162:Transfection; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D003574:Cytochrome c Group; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein