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 membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax 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 with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP-Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.
Wolter
K G
KG
Biochemistry Section, Surgical Neurology Branch, Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hsu
Y T
YT
Smith
C L
CL
Nechushtan
A
A
Xi
X G
XG
Youle
R J
RJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001692:Biological Transport; D019556:COS Cells; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism*; D004058:Diffusion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D049452:Green Fluorescent Proteins