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 Bar may regulat e apoptosis. The spatial and temporal interaction of Bcl-2 and Bar at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance e nergy transfer). Using green fluorescent protein (GFP)-Bax and blue fl uorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bar in ind ividual mitochondria. Mitochondrially localized cytochrome c-GFP and B FP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human p apillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in th e 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.