Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation.
Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-deltaEEVD) or the ATP hydrolytic domain (hsp70-deltaATPase) failed to prevent mitochondrial AIF release. Although hsp70-deltaEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-deltaATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.
Ruchalski
Kathleen
K
Renal Section, Department of Medicine, Boston Medical Center, Boston University, Boston, Massachusetts 02118-2518, USA.
Mao
Haiping
H
Li
Zhijian
Z
Wang
Zhiyong
Z
Gillers
Sara
S
Wang
Yihan
Y
Mosser
Dick D
DD
Gabai
Vladimir
V
Schwartz
John H
JH
Borkan
Steven C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D017353:Gene Deletion; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D047468:Immunoprecipitation; D007668:Kidney; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D015233:Models, Statistical; D018832:Molecular Chaperones / Q000378:metabolism; D009893:Opossums; D017434:Protein Structure, Tertiary; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D051033:Apoptosis Inducing Factor; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004229:Dithiothreitol