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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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Monomeric (MutM) mutants of Beclin 1 CC domain retain interaction with Atg14L and UVRAG.(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
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f5: Monomeric (MutM) mutants of Beclin 1 CC domain retain interaction with Atg14L and UVRAG.(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).

Mentions: We further investigated the interaction of Beclin 1 MutM mutants with Atg14L and UVRAG in cultured cells. We expressed GFP–Beclin 1 variants (including wild-type and MutM mutations) alone or together with FLAG-UVRAG in HEK 293T cells and assessed by co-IP assay the interaction of Beclin 1 with endogenous Atg14L (Fig. 5a) or FLAG-UVRAG (Fig. 5b), respectively. The wild-type GFP–Beclin 1 and MutMs L178A/L192A, L178A/L196A and L178A/L259A co-IP in a similar efficiency with endogenous Atg14L or FLAG-UVRAG. Taken together, the result demonstrates that the Beclin 1 mutants defective in homodimer formation are not affected in Beclin 1–Atg14L or Beclin 1–UVRAG heterodimer formation. Therefore, the monomeric form of Beclin 1 is sufficient for interaction with Atg14L and UVRAG.


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)

Monomeric (MutM) mutants of Beclin 1 CC domain retain interaction with Atg14L and UVRAG.(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293417&req=5

f5: Monomeric (MutM) mutants of Beclin 1 CC domain retain interaction with Atg14L and UVRAG.(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
Mentions: We further investigated the interaction of Beclin 1 MutM mutants with Atg14L and UVRAG in cultured cells. We expressed GFP–Beclin 1 variants (including wild-type and MutM mutations) alone or together with FLAG-UVRAG in HEK 293T cells and assessed by co-IP assay the interaction of Beclin 1 with endogenous Atg14L (Fig. 5a) or FLAG-UVRAG (Fig. 5b), respectively. The wild-type GFP–Beclin 1 and MutMs L178A/L192A, L178A/L196A and L178A/L259A co-IP in a similar efficiency with endogenous Atg14L or FLAG-UVRAG. Taken together, the result demonstrates that the Beclin 1 mutants defective in homodimer formation are not affected in Beclin 1–Atg14L or Beclin 1–UVRAG heterodimer formation. Therefore, the monomeric form of Beclin 1 is sufficient for interaction with Atg14L and UVRAG.

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