Limits...
Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

Baskaran S, Carlson LA, Stjepanovic G, Young LN, Kim do J, Grob P, Stanley RE, Nogales E, Hurley JH - Elife (2014)

Bottom Line: Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V.The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point.These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Show MeSH

Related in: MedlinePlus

MBP-tagging identifies PI3KC3-C1 subunits.(A) Six different MBP-tagged versions of the complex were used to identify the position of the different PI3KC3-C1 subunits. A cartoon diagram indicating the position of the MBP tags is shown on the right. The two left columns show the reference-free 2D class averages of the MBP-labeled samples and arrowhead highlights the MBP tag position. The middle column shows the corresponding class for the unlabeled sample. Two right columns show the difference map calculated by subtracting the unlabeled reference class from the labeled and the dotted circle represents the MBP density. (B) 3D reconstruction of the PI3KC3-C1 complex highlighting the position of the six MBP tags used for domain mapping.DOI:http://dx.doi.org/10.7554/eLife.05115.006
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4281882&req=5

fig4: MBP-tagging identifies PI3KC3-C1 subunits.(A) Six different MBP-tagged versions of the complex were used to identify the position of the different PI3KC3-C1 subunits. A cartoon diagram indicating the position of the MBP tags is shown on the right. The two left columns show the reference-free 2D class averages of the MBP-labeled samples and arrowhead highlights the MBP tag position. The middle column shows the corresponding class for the unlabeled sample. Two right columns show the difference map calculated by subtracting the unlabeled reference class from the labeled and the dotted circle represents the MBP density. (B) 3D reconstruction of the PI3KC3-C1 complex highlighting the position of the six MBP tags used for domain mapping.DOI:http://dx.doi.org/10.7554/eLife.05115.006

Mentions: The 28 Å reconstruction described above lacked the detail, on its own, to unambiguously place most of the structures of the various subunit domains. The exceptions were the donut-shaped region corresponding to the WD40 domain of VPS15 and the arch-shaped density at the base of the V corresponding to the HEAT repeat of the same subunit. Therefore, we generated and purified a series of N- and C-terminal MBP fusion constructs to map the domains using EM (Figure 4A,B). Both N- and C-terminal MBP fusions of VPS34 and BECN1 and the N-terminal MBP fusions of ATG14 and VPS15 were successfully purified and imaged.10.7554/eLife.05115.006Figure 4.MBP-tagging identifies PI3KC3-C1 subunits.


Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

Baskaran S, Carlson LA, Stjepanovic G, Young LN, Kim do J, Grob P, Stanley RE, Nogales E, Hurley JH - Elife (2014)

MBP-tagging identifies PI3KC3-C1 subunits.(A) Six different MBP-tagged versions of the complex were used to identify the position of the different PI3KC3-C1 subunits. A cartoon diagram indicating the position of the MBP tags is shown on the right. The two left columns show the reference-free 2D class averages of the MBP-labeled samples and arrowhead highlights the MBP tag position. The middle column shows the corresponding class for the unlabeled sample. Two right columns show the difference map calculated by subtracting the unlabeled reference class from the labeled and the dotted circle represents the MBP density. (B) 3D reconstruction of the PI3KC3-C1 complex highlighting the position of the six MBP tags used for domain mapping.DOI:http://dx.doi.org/10.7554/eLife.05115.006
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281882&req=5

fig4: MBP-tagging identifies PI3KC3-C1 subunits.(A) Six different MBP-tagged versions of the complex were used to identify the position of the different PI3KC3-C1 subunits. A cartoon diagram indicating the position of the MBP tags is shown on the right. The two left columns show the reference-free 2D class averages of the MBP-labeled samples and arrowhead highlights the MBP tag position. The middle column shows the corresponding class for the unlabeled sample. Two right columns show the difference map calculated by subtracting the unlabeled reference class from the labeled and the dotted circle represents the MBP density. (B) 3D reconstruction of the PI3KC3-C1 complex highlighting the position of the six MBP tags used for domain mapping.DOI:http://dx.doi.org/10.7554/eLife.05115.006
Mentions: The 28 Å reconstruction described above lacked the detail, on its own, to unambiguously place most of the structures of the various subunit domains. The exceptions were the donut-shaped region corresponding to the WD40 domain of VPS15 and the arch-shaped density at the base of the V corresponding to the HEAT repeat of the same subunit. Therefore, we generated and purified a series of N- and C-terminal MBP fusion constructs to map the domains using EM (Figure 4A,B). Both N- and C-terminal MBP fusions of VPS34 and BECN1 and the N-terminal MBP fusions of ATG14 and VPS15 were successfully purified and imaged.10.7554/eLife.05115.006Figure 4.MBP-tagging identifies PI3KC3-C1 subunits.

Bottom Line: Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V.The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point.These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Show MeSH
Related in: MedlinePlus