Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer.
It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.
Mahajan
N P
NP
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599, USA.
Linder
K
K
Berry
G
G
Gordon
G W
GW
Heim
R
R
Herman
B
B
D016475:3T3 Cells; D000818:Animals; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015335:Molecular Probes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins