rab5a  (Jena Bioscience)

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Schematic illustration of the Rab5-VPS34CII-PI3P-PX signaling pathway. Shown is a schematic view of the Rab5-VPS34CII-PI3P-PX pathway that assembles on phagosome and early endosomal membranes, where the small G protein GTP-Rab5A (Rab5) regulates the lipid kinase VPS34CII via an unknown mechanism explored herein. The four subunits of VPS34CII are VPS34 (blue), VPS15 (rose), Beclin1 (gold), and UVRAG (green). VPS34CII phosphorylates the substrate lipid phosphatidylinositol (PI) to generate the signaling lipid PI-3-phosphate (PI3P). The resulting PI3P recruits multiple signaling proteins possessing a PI3P-specific membrane targeting domain such as FYVE, PX (shown), or PROPPINS to initiate key cellular processes. In the single-molecule membrane density and kinase activity assays, an Alexa-Fluor-labeled (gold symbol) nanobody or PX domain sensor is used to detect and count membrane-bound VPS34CII or PI3P product molecules, respectively. In addition, the reconstituted system employs an engineered Rab5 anchored to the membrane by reaction of native lipidation residue Cys212 (the more N-terminal site of the two native lipidation sites shown in the figure) with the maleimide headgroup of lipid PE-Mal. The illustrated structural model of human VPS34CII is based on the yeast VPS34 structure (Protein Data Bank, PDB: 5DFZ (23)) and on a recently reported model of membrane-bound human VPS34CII (12). The structure of the Rab5-VPS34CII complex is not yet known but is believed to involve an Rab5-VPS15(WD40) contact as indicated (4,10,11). The PX domain is from P40phox (PDB: 1H6H (51)). Shown are structures generated in MacPyMol. To see this figure in color, go online.

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Activity Assay, Labeling, Generated

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Effect of membrane-anchored Rab5A on net VPS34CII lipid kinase activity: superactivating conditions. (A) Single-molecule TIRFM images show VPS34CII lipid kinase reactions initiated at t = 0 while monitoring the product lipid PI3P using a saturating level of fluor-labeled PX domain to bind and detect individual product PI3P molecules. The findings show that under superactivating conditions, VPS34CII lipid kinase activity can be detected in the absence of Rab5 but increases dramatically in the presence of membrane-anchored GTP-Rab5. (B) Shown is the single-molecule time course of the VPS34CII lipid kinase reaction plotting the number of product molecules generated per field as a function of reaction time, illustrating stimulation by membrane-anchored GTP-Rab5. (C) Given is the rate of the VPS34CII lipid kinase reaction defined by the slope of the single-molecule time course (previous panel), illustrating the significant, 33 ± 12-fold (p < 0.001) rate enhancement by membrane-anchored GTP-loaded Rab5 ((+) GTP-Rab5) relative to membranes lacking the G protein ((−) GTP-Rab5). Also shown are controls demonstrating the lack of stimulation by membrane-anchored, GDP-loaded Rab5 ((+) GDP-Rab5) or by membranes pretreated with a maleimide quencher to block GTP-Rab5 anchoring to the membrane ((+) quenched Rab5) before the anchoring reaction and washing to remove free GTP-Rab5. Single-molecule kinase reactions under were carried out at 21 ± 0.5°C under superactivating conditions: the supported lipid bilayer PC/PE/PS/PI/Mal-PE was 49:20:25:5:1 mol %, and reaction buffer 25 mM HEPES (pH 8.0), 150 mM NaCl, 5 mM glutathione, 2.5 mM EGTA, 5 mM Mn2+, 1 mM GTP, and 1 mM ATP. The observation field was 60 × 60 μm2.

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Activity Assay, Labeling, Generated, Blocking Assay

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Effect of membrane-anchored GTP-Rab5A on VPS34CII membrane surface density: superactivating conditions. (A) Shown is the single-molecule surface density of VPS34CII molecules stably bound to the target supported lipid bilayer, showing the significant, 6.6 ± 2.5-fold (p < 0.001) enhancement of VPS34CII density on membranes possessing anchored GTP-Rab5A relative to membranes lacking Rab5 (338.0 ± 52 and 51.6 ± 28 molecules field−1, respectively). Single VPS34CII molecules were detected and counted using a fluorescent, monoclonal sensor nanobody to tag individual kinase molecules. (B) Controls were carried out to ascertain whether the sensor nanobody perturbed the membrane binding or lipid kinase activities of VPS34CII. Shown are single-molecule kinase assay data (Fig. 4) revealing that nanobody has no significant effect, within error, on the net rate of VPS34CII production of PI3P either in the absence or presence of membrane-anchored GTP-Rab5A. These findings provide strong evidence that nanobody binding has a negligible effect on VPS34CII membrane association and specific kinase activity, consistent with the HDX-MS data, revealing that the nanobody docking surface is distal from the kinase membrane docking and active sites (Fig. S1; Table 1). For both panels, superactivating conditions is the same as above (see Fig. 4 legend). The observation field was 60 × 60 μm2.

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Stable Transfection, Binding Assay, Kinase Assay, Activity Assay

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Effect of membrane-anchored GTP-Rab5A on the kinetics of VPS34CII membrane association and dissociation: superactivating conditions. (A) The membrane-anchored GTP-Rab5 significantly increases by 3.6 ± 1.5-fold (p = 0.01), the pseudo-first-order, on-rate constant (k’on) for the appearance of stably bound VPS34CII single molecules on the target membrane surface, from k’on = 4.7 × 106 ± 1.6 × 106 events per (μm2 × [VPS34CII, M] × s) in the absence of Rab5 to 1.7 × 107 ± 4.2 × 106 events per (μm2 × [VPS34CII, M] × s) in the presence of anchored GTP-Rab5. (B) The membrane-anchored GTP-Rab5 significantly decreases by 1.9 ± 0.1-fold (p = 0.002), the first-order off-rate constant for VPS34CII membrane dissociation (koff), from 4.2 ± 0.3 s−1 in the absence of Rab5A to 8.2 ± 0.7 s−1 in the presence of anchored GTP-Rab5. Dissociation rates were obtained from the indicated bound state lifetime distributions for populations of single VPS34CII molecules. In both experiments, single molecules of VPS34CII were detected by a fluorescent nanobody sensor under superactivating conditions as above (see Figs. 4 and ​and55 legends).

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Stable Transfection

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Effect of membrane-anchored GTP-Rab5A on the specific activity (turnover rate) of membrane-bound VPS34CII: superactivating conditions. (A) The membrane-anchored GTP-Rab5 significantly increases by 5.2 ± 1.8-fold (p = 0.003), the turnover rate of the average membrane-bound VPS34CII molecule from 27 ± 8 PI3P products min−1 in the absence of Rab5A to 141 ± 32 PI3P products min−1 in the presence of anchored GTP-Rab5A. The superactivating conditions are the same as above (see Figs. 4 and ​and55 legends).

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Activity Assay

Journal: Biophysical Journal

Article Title: The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

doi: 10.1016/j.bpj.2020.10.028

Figure Lengend Snippet: Subunit and domain organization of proteins employed in this study. (A) Human VPS34CII is a class III PI3K composed of four multidomain subunits as shown. The protein employed in this study is the full-length, tag-less heterotetramer purified from mammalian cells via a double protein A purification tag (ZZ tag) at the C-terminus of VPS15 that was proteolytically removed by TEV protease to give the construct shown. (B) Human Rab5A (Rab5) is a small, monomeric G protein. The construct employed herein is purified from bacterial cells via its 6×-His purification tag and then loaded with GTP. The construct possesses an active site mutation (Q79L) that blocks GTP hydrolysis and retains a single surface Cys residue for membrane anchoring at position 212. The C212 residue near the native C-terminus is a native lipidation site, and in this construct, C212 is placed at the C-terminus by truncation Δ213–215, whereas the 6×-His tag is removed by SUMO proteolysis during purification. (C) The isolated PX domain of human P40phox is purified from bacterial cells via its GST purification tag. The construct is mutated to possess a single-surface Cys residue near the N-terminus for labeling with fluorophore for use as a sensor for its target PI3P lipid. The GST tag is removed by proteolysis during purification via TEV proteolysis. See Materials and Methods and (12).

Article Snippet: To exchange Rab5A into the desired nucleotide, ∼11-fold excess GTP or GDP (Jena Bioscience, Jena, Germany) was added to the concentrate as well as 10 mM ethylenediaminetetraacetic acid and allowed to incubate for 1.5 h at room temperature.

Techniques: Purification, Construct, Mutagenesis, Isolation, Labeling