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Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG.

Li X, He L, Che KH, Funderburk SF, Pan L, Pan N, Zhang M, Yue Z, Zhao Y - Nat Commun (2012)

Bottom Line: The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity.Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly.These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology and Chemical Technology, State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR China.

ABSTRACT
Beclin 1 is a core component of the Class III Phosphatidylinositol 3-Kinase VPS34 complex. The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity. Here we report the crystal structure of the coiled coil domain that forms an antiparallel dimer and is rendered metastable by a series of 'imperfect' a-d' pairings at its coiled coil interface. Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly. Beclin 1 mutants with their 'imperfect' a-d' pairings modified to enhance self-interaction, show distinctively altered interactions with Atg14L or UVRAG. These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

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Subcellular localization of Beclin 1 MutM and MutStab with co-expressed Atg14L or UVRAG proteins and early autophagosome markers.(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed–Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed–Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta. (c–e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed–Atg14L (red) and GFP-tagged Atg12 (c), LC3 (d) and DFCP (e) (green). The scale bar is 20 μM. (f). A proposed model for the dynamic transition among homodimer (antiparallel) of Beclin 1, and the heterodimers of Beclin 1–Atg14L and Beclin 1–UVRAG. Multiple homo- and hetero-oligomeric forms of Beclin 1 protein exist intracellularly, and the nature of metastability in Beclin 1 CCD interface facilitates the transition of Beclin 1 homodimer to Beclin 1–Atg14L/UVRAG heterodimer that are compatible with activated autophagy-lysosomal degradation. The star shapes indicate the imperfect dimer interface. The thicker arrows pointing towards Beclin 1–UVRAG complex indicate its stronger affinity.
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f7: Subcellular localization of Beclin 1 MutM and MutStab with co-expressed Atg14L or UVRAG proteins and early autophagosome markers.(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed–Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed–Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta. (c–e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed–Atg14L (red) and GFP-tagged Atg12 (c), LC3 (d) and DFCP (e) (green). The scale bar is 20 μM. (f). A proposed model for the dynamic transition among homodimer (antiparallel) of Beclin 1, and the heterodimers of Beclin 1–Atg14L and Beclin 1–UVRAG. Multiple homo- and hetero-oligomeric forms of Beclin 1 protein exist intracellularly, and the nature of metastability in Beclin 1 CCD interface facilitates the transition of Beclin 1 homodimer to Beclin 1–Atg14L/UVRAG heterodimer that are compatible with activated autophagy-lysosomal degradation. The star shapes indicate the imperfect dimer interface. The thicker arrows pointing towards Beclin 1–UVRAG complex indicate its stronger affinity.

Mentions: In a previous study, we showed that simultaneous overexpression of Beclin 1 and Atg14L in transfected cells promoted the co-localized Beclin 1–Atg14L puncta and recruited early autophagosomes marker19. Using this assay, we examined the localization of co-expressed AsRed–Atg14L and Beclin 1–GFP variants. As expected, AsRed–Atg14L and wild-type Beclin 1–GFP co-localized in intracellular discrete puncta. Similar to wild-type Beclin 1, the GFP–Beclin 1 MutM L178A/L192A also formed puncta with AsRed–Atg14L. By contrast, GFP–Beclin 1 MutStabs, E189L/A255L and E189L/A217L/E224L/A255L, are largely diffused when co-expressed with AsRed–Atg14L (Fig. 7a). This result indicates that Beclin 1 MutMs retain the ability to bind Atg14L and promote autophagosome formation, whereas Beclin 1 MutStabs are defective in interaction with Atg14L, and perhaps impaired in autophagy regulation. However, co-expression of UVRAG and Beclin 1 variants (including Beclin1 wild type, all MutM and MutStab mutants) led to diffused fluorescent signalling throughout cytoplasm (Fig. 7b). Taken together, the results suggest that the Beclin 1–Atg14L interaction is highly sensitive to the metastable imperfect dimer interface of Beclin 1. The monomeric form (MutMs) with the imperfect residues at the dimer interface retained co-localize with Atg14L. In contrast the stabilized dimeric form (MutStabs) with the imperfect residues altered can not. In contrast, the Beclin 1–UVRAG interaction is less sensitive to the imperfect dimer interface of Beclin 1 and seems to have a cellular localization profile distinct from that of the Beclin 1–Atg14L interaction.


Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG.

Li X, He L, Che KH, Funderburk SF, Pan L, Pan N, Zhang M, Yue Z, Zhao Y - Nat Commun (2012)

Subcellular localization of Beclin 1 MutM and MutStab with co-expressed Atg14L or UVRAG proteins and early autophagosome markers.(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed–Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed–Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta. (c–e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed–Atg14L (red) and GFP-tagged Atg12 (c), LC3 (d) and DFCP (e) (green). The scale bar is 20 μM. (f). A proposed model for the dynamic transition among homodimer (antiparallel) of Beclin 1, and the heterodimers of Beclin 1–Atg14L and Beclin 1–UVRAG. Multiple homo- and hetero-oligomeric forms of Beclin 1 protein exist intracellularly, and the nature of metastability in Beclin 1 CCD interface facilitates the transition of Beclin 1 homodimer to Beclin 1–Atg14L/UVRAG heterodimer that are compatible with activated autophagy-lysosomal degradation. The star shapes indicate the imperfect dimer interface. The thicker arrows pointing towards Beclin 1–UVRAG complex indicate its stronger affinity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: Subcellular localization of Beclin 1 MutM and MutStab with co-expressed Atg14L or UVRAG proteins and early autophagosome markers.(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed–Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed–Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta. (c–e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed–Atg14L (red) and GFP-tagged Atg12 (c), LC3 (d) and DFCP (e) (green). The scale bar is 20 μM. (f). A proposed model for the dynamic transition among homodimer (antiparallel) of Beclin 1, and the heterodimers of Beclin 1–Atg14L and Beclin 1–UVRAG. Multiple homo- and hetero-oligomeric forms of Beclin 1 protein exist intracellularly, and the nature of metastability in Beclin 1 CCD interface facilitates the transition of Beclin 1 homodimer to Beclin 1–Atg14L/UVRAG heterodimer that are compatible with activated autophagy-lysosomal degradation. The star shapes indicate the imperfect dimer interface. The thicker arrows pointing towards Beclin 1–UVRAG complex indicate its stronger affinity.
Mentions: In a previous study, we showed that simultaneous overexpression of Beclin 1 and Atg14L in transfected cells promoted the co-localized Beclin 1–Atg14L puncta and recruited early autophagosomes marker19. Using this assay, we examined the localization of co-expressed AsRed–Atg14L and Beclin 1–GFP variants. As expected, AsRed–Atg14L and wild-type Beclin 1–GFP co-localized in intracellular discrete puncta. Similar to wild-type Beclin 1, the GFP–Beclin 1 MutM L178A/L192A also formed puncta with AsRed–Atg14L. By contrast, GFP–Beclin 1 MutStabs, E189L/A255L and E189L/A217L/E224L/A255L, are largely diffused when co-expressed with AsRed–Atg14L (Fig. 7a). This result indicates that Beclin 1 MutMs retain the ability to bind Atg14L and promote autophagosome formation, whereas Beclin 1 MutStabs are defective in interaction with Atg14L, and perhaps impaired in autophagy regulation. However, co-expression of UVRAG and Beclin 1 variants (including Beclin1 wild type, all MutM and MutStab mutants) led to diffused fluorescent signalling throughout cytoplasm (Fig. 7b). Taken together, the results suggest that the Beclin 1–Atg14L interaction is highly sensitive to the metastable imperfect dimer interface of Beclin 1. The monomeric form (MutMs) with the imperfect residues at the dimer interface retained co-localize with Atg14L. In contrast the stabilized dimeric form (MutStabs) with the imperfect residues altered can not. In contrast, the Beclin 1–UVRAG interaction is less sensitive to the imperfect dimer interface of Beclin 1 and seems to have a cellular localization profile distinct from that of the Beclin 1–Atg14L interaction.

Bottom Line: The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity.Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly.These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology and Chemical Technology, State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR China.

ABSTRACT
Beclin 1 is a core component of the Class III Phosphatidylinositol 3-Kinase VPS34 complex. The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity. Here we report the crystal structure of the coiled coil domain that forms an antiparallel dimer and is rendered metastable by a series of 'imperfect' a-d' pairings at its coiled coil interface. Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly. Beclin 1 mutants with their 'imperfect' a-d' pairings modified to enhance self-interaction, show distinctively altered interactions with Atg14L or UVRAG. These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

Show MeSH
Related in: MedlinePlus