regulation of protein serine/threonine kinase activity / lytic vacuole / maintenance of Golgi location / regulation of endosome size / nucleus-vacuole junction / cellular response to aluminum ion / cytoplasmic side of early endosome membrane / positive regulation of protein lipidation / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade ...regulation of protein serine/threonine kinase activity / lytic vacuole / maintenance of Golgi location / regulation of endosome size / nucleus-vacuole junction / cellular response to aluminum ion / cytoplasmic side of early endosome membrane / positive regulation of protein lipidation / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / Synthesis of PIPs at the late endosome membrane / positive regulation of stress granule assembly / phosphatidylinositol 3-kinase complex, class III / cellular response to oxygen-glucose deprivation / Synthesis of PIPs at the early endosome membrane / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / synaptic vesicle recycling / host-mediated activation of viral genome replication / response to mitochondrial depolarisation / amyloid-beta clearance by transcytosis / SARS-CoV-2 modulates autophagy / positive regulation of attachment of mitotic spindle microtubules to kinetochore / presynaptic endosome / negative regulation of lysosome organization / engulfment of apoptotic cell / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol kinase activity / host-mediated perturbation of viral process / positive regulation of autophagosome assembly / response to L-leucine / early endosome to late endosome transport / regulation of filopodium assembly / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / protein localization to phagophore assembly site / multivesicular body sorting pathway / receptor catabolic process / protein targeting to vacuole / RAB geranylgeranylation / protein targeting to lysosome / endosome organization / SMAD protein signal transduction / regulation of autophagosome assembly / late endosome to vacuole transport / pexophagy / RAB GEFs exchange GTP for GDP on RABs / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / early phagosome / double-strand break repair via classical nonhomologous end joining / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / centrosome cycle / positive regulation of autophagosome maturation / phosphatidylinositol-3-phosphate biosynthetic process / TBC/RABGAPs / cellular response to nitrogen starvation / negative regulation of programmed cell death / SNARE complex assembly / spindle organization / phosphatidylinositol 3-kinase / lysosome organization / 1-phosphatidylinositol-3-kinase activity / response to iron(II) ion / mitotic metaphase chromosome alignment / response to vitamin E / cytoplasmic pattern recognition receptor signaling pathway / Macroautophagy / positive regulation of cardiac muscle hypertrophy / phosphatidylinositol-mediated signaling / RSV-host interactions / p38MAPK cascade / regulation of synaptic vesicle exocytosis / phosphatidylinositol phosphate biosynthetic process / Synthesis of PIPs at the plasma membrane / autolysosome / positive regulation of exocytosis / Respiratory syncytial virus (RSV) attachment and entry / autophagosome membrane / synaptic vesicle endocytosis / PI3K Cascade / axoneme / RHO GTPases Activate NADPH Oxidases / autophagosome assembly / chromosome, centromeric region / endocytic vesicle / autophagosome maturation / amyloid-beta metabolic process / regulation of macroautophagy / canonical Wnt signaling pathway / phagocytosis / neuron development / cellular defense response / intercellular bridge / cellular response to glucose starvation / phosphatidylinositol 3-kinase binding / mitophagy / positive regulation of intrinsic apoptotic signaling pathway / cellular response to copper ion / phagocytic vesicle 類似検索 - 分子機能
UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / : / VPS15-like, helical domain / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain ...UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / : / VPS15-like, helical domain / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain / Atg6/beclin, coiled-coil domain / Apg6 BARA domain / Apg6 coiled-coil region / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / Small GTPase Rab domain profile. / C2 phosphatidylinositol 3-kinase-type domain / Phosphoinositide 3-kinase C2 / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, region postulated to contain C2 domain / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / C2 domain superfamily / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / WD domain, G-beta repeat / Armadillo-type fold / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase 類似検索 - ドメイン・相同性
GUANOSINE-5'-TRIPHOSPHATE / Ras-related protein Rab-5A / Beclin-1 / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4 / UV radiation resistance-associated gene protein 類似検索 - 構成要素
ジャーナル: Nat Commun / 年: 2021 タイトル: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes. 著者: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel / ...著者: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel / Juri Rappsilber / Kathrin Lang / Sean Munro / John A G Briggs / Roger L Williams / 要旨: The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal ...The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
COMPLEX
The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 (encoding UVGRAG 1-464 fused to the BATS of ATG14, residues 413-492 in pVAG) and pYO1006 (Beclin1 in pCAG)
#1-#6
0
MULTIPLESOURCES
2
human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
COMPLEX
#1-#5
1
RECOMBINANT
3
Ras-related protein Rab-5A
COMPLEX
#6
1
RECOMBINANT
分子量
値: 392615 kDa/nm / 実験値: NO
由来(天然)
ID
Entity assembly-ID
生物種
Ncbi tax-ID
1
2
Homo sapiens (ヒト)
9606
2
3
Homo sapiens (ヒト)
9606
由来(組換発現)
ID
Entity assembly-ID
生物種
Ncbi tax-ID
1
2
Homo sapiens (ヒト)
9606
2
3
Escherichia coli str. K-12 substr. DH10B (大腸菌)
316385
緩衝液
pH: 8
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
50mM
HEPES
HEPES
1
2
200mM
NaCl
NaCl
1
3
0.5mM
TCEP
TCEP
1
試料
濃度: 6.2 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
used external restraints where high-resolution homologues were available
15
ISOLDE
1.1
モデル精密化
MDFlexiblefit
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
対称性
点対称性: C1 (非対称)
3次元再構成
解像度: 9.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 26979 / アルゴリズム: FOURIER SPACE / 対称性のタイプ: POINT
EM volume selection
Num. of tomograms: 105 / Num. of volumes extracted: 191196 / Reference model: two Gaussian filtered elipsoids
原子モデル構築
B value: 320 / プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: correlation 詳細: An initial model was build with SWISSMODEL. using 5DFZ, 4DDP and 3IHY. This was then fit manually to density while running restrained MD with ISOLDE. The model was refined in REFMAC with self- ...詳細: An initial model was build with SWISSMODEL. using 5DFZ, 4DDP and 3IHY. This was then fit manually to density while running restrained MD with ISOLDE. The model was refined in REFMAC with self-restraints (4.3) and restraints to 3MJH, 4DDP and 3IHY.
原子モデル構築
3D fitting-ID: 1 / Source name: PDB / タイプ: experimental model
解像度: 9.8→234.63 Å / Cor.coef. Fo:Fc: 0.954 / WRfactor Rwork: 0.1573 / SU B: 303.369 / SU ML: 1.88 / Average fsc overall: 0.9814 / Average fsc work: 0.9814 詳細: Hydrogens have been used if present in the input file
Rfactor
反射数
%反射
Rwork
0.1573
11064
-
all
0.1573
-
-
Rfree
-
-
0 %
obs
-
-
100 %
溶媒の処理
溶媒モデル: BABINET MODEL
原子変位パラメータ
Biso mean: 320.935 Å2
Baniso -1
Baniso -2
Baniso -3
1-
-6.184 Å2
1.147 Å2
-0.935 Å2
2-
-
-1.872 Å2
5.156 Å2
3-
-
-
8.056 Å2
拘束条件
Refine-ID
タイプ
Dev ideal
Dev ideal target
数
ELECTRONMICROSCOPY
r_bond_refined_d
0.007
0.011
15196
ELECTRONMICROSCOPY
r_ext_dist_refined_d
0.003
0.1
191935
ELECTRONMICROSCOPY
r_angle_refined_deg
1.977
1.633
21206
ELECTRONMICROSCOPY
r_dihedral_angle_1_deg
9.059
5
2881
ELECTRONMICROSCOPY
r_dihedral_angle_2_deg
33.74
23.676
68
ELECTRONMICROSCOPY
r_dihedral_angle_3_deg
16.618
15
241
ELECTRONMICROSCOPY
r_dihedral_angle_4_deg
17.49
15
6
ELECTRONMICROSCOPY
r_chiral_restr
0.126
0.2
2778
ELECTRONMICROSCOPY
r_gen_planes_refined
0.01
0.02
12322
ELECTRONMICROSCOPY
r_nbd_refined
0.236
0.2
12534
ELECTRONMICROSCOPY
r_nbtor_refined
0.266
0.2
21122
ELECTRONMICROSCOPY
r_xyhbond_nbd_refined
0.081
0.2
742
ELECTRONMICROSCOPY
r_mcbond_it
78.101
49.729
11560
ELECTRONMICROSCOPY
r_mcangle_it
136.303
74.158
14429
ELECTRONMICROSCOPY
r_scbond_it
86.101
48.866
3636
ELECTRONMICROSCOPY
r_scangle_it
135.451
73.092
6777
ELECTRONMICROSCOPY
r_lrange_it
264.032
1652.509
192054
LS精密化 シェル
Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %