Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor.
Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150-268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFDelta150-268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF-HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.
Gurbuxani
Sandeep
S
INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France.
Schmitt
Elise
E
Cande
Celine
C
Parcellier
Arnaud
A
Hammann
Arlette
A
Daugas
Eric
E
Kouranti
Ilektra
I
Spahr
Chris
C
Pance
Alena
A
Kroemer
Guido
G
Garrido
Carmen
C
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis; D051033:Apoptosis Inducing Factor; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions
D051033:Apoptosis Inducing Factor; D018076:DNA, Complementary; D005420:Flavoproteins; D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C494830:AIFM1 protein, mouse; D010455:Peptides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins