Abstract
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.
Highlights
Apoptosis or programmed cell death is responsible for the removal of unwanted or supernumerary cells during development, as well as in adult homeostasis (JacobsonAnother apoptogenic molecule released from the mitochondria is the flavoprotein apoptosis-inducing factor (AIF)
We have previously shown by coimmunoprecipitation studies that AIF interacted with Heat shock protein 70 (HSP70), but not with HSP90, HSP60 or HSP27 (Ravagnan et al, 2001)
In order to evaluate the physiological relevance of this association, we studied whether HSP70 interacted with AIF in vivo
Summary
Apoptosis or programmed cell death is responsible for the removal of unwanted or supernumerary cells during development, as well as in adult homeostasis Another apoptogenic molecule released from the mitochondria is the flavoprotein apoptosis-inducing factor (AIF). AIF released into the cytosol is imported into the nucleus, where it triggers caspase-independent nuclear changes (Susin et al, 1999; Daugas et al, 2000). Mature AIF is a flavoprotein with weak but significant sequence homology with bacterial oxidoreductases, suggesting that it is a bifunctional protein with a mitochondrial-resident role and an apoptogenic function. Since the flavin adenine dinucleotide (FAD) cofactor is dispensable for the apoptogenic function, but required for the oxidoreductase activity of AIF (Susin et al, 1999; Miramar et al, 2001), the structural
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