A cytochrome c–GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells

• Published in: FEBS letters Vol. 471; no. 2; pp. 235 - 239
• Main Authors: Roucou, Xavier, Prescott, Mark, Devenish, Rodney J, Nagley, Phillip
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 14.04.2000
• Subjects: Animals; Apoptosis - drug effects; Bax; bcl-2-Associated X Protein; Biological Transport - drug effects; Blotting, Western; Cell death; Cytochrome c; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Cytochrome c Group - metabolism; Cytochromes c; Cytoplasm - drug effects; Cytoplasm - enzymology; Cytoplasm - metabolism; Doxycycline - pharmacology; Electron Transport; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Deletion; Gene Expression - drug effects; Green fluorescent protein; Green Fluorescent Proteins; Intracellular Membranes - chemistry; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Lasers; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microscopy, Confocal; Mitochondria - chemistry; Mitochondria - drug effects; Mitochondria - enzymology; Mitochondria - metabolism; Mitochondrion; Molecular Weight; Nystatin - pharmacology; Permeability - drug effects; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Spheroplasts - cytology; Spheroplasts - drug effects; Spheroplasts - genetics; Spheroplasts - metabolism; Time Factors; Yeast; Cytochrome c; Mitochondrion; Bax; Yeast; Cell death; Green fluorescent protein
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)01404-6

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c–GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c–GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c–GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c–GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c–GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.