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Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.

Abstract：

:Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Mei Y,Su M,Sanishvili R,Chakravarthy S,Colbert CL,Sinha SC

doi

10.1021/acs.biochem.6b00246

subject

Has Abstract

pub_date

2016-08-02 00:00:00

pages

4239-53

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.