MOVEMENT OF BAX FROM THE CYTOSOL TO MITOCHONDRIA DURING APOPTOSIS

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral m embrane protein associated with organelles or bound to or ganelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 fami ly member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-X-L. Confoca l microscopy performed on living Cos-7 kidney epithelial cells and L92 9 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-X-L had a punctate d istribution and colocalized with a mitochondrial marker, whereas GFP-B ax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble protein, in contrast to organelle -bound GFP-Bcl-2. The diffuse localization of GFP-Bax did not change w ith coexpression of high levels of Bcl-2 or Bcl-X-L. However, upon ind uction of apoptosis, GFP-Bax moved intracellularly to a punctate distr ibution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkag e or nuclear condensation. Removal of a COOH-terminal hydrophobic doma in from GFP-Bax inhibited redistribution during apoptosis and inhibite d the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic cha nge occurs in the intracellular localization of Bax, and this redistri bution of soluble Bax to organelles appears important for Bax to promo te cell death.