Defining the subcellular distribution and metabolic channeling of phosphatidylinositol

Abstract

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

Figure S1.

Structural features of the BcPI-PLC scaffold inform the rationale design of the BcPI-PLC^H82A substrate trap. (A) Structural comparison of the L. monocytogenes (top row, left, beige; PDB accession no. 1AOD) and B. cereus (top row, center, blue and yellow; PDB accession no. 1PTG) PI-PLCs bound to myo-inositol. Notable differences on the membrane-oriented surface, including a general expansion of the PI-binding pocket in the BcPI-PLC, are highlighted in yellow and presented on a surface rendering of the binding pocket (top row, right). Ribbon representations of the aligned structures are shown on the bottom right alongside representative images of HEK293-AT1 cells expressing either the L. monocytogenes mRFP–PI-PLC (bottom row, left) or EGFP–BcPI-PLC (bottom row, center; scale bars, 10 µm). (B) Electron density map (Fo-Fc, 3.0σ) surrounding the myo-inositol headgroup within the BcPI-PLC^H82A active site shows the intact coordination of the inositol ring. (C) Table listing statistics used for crystallographic data collection and refinement, which are also indexed in the associated PDB entry (accession no. 6S2A).

Figure 1.

Visualizing the subcellular distribution of PI using the BcPI-PLC scaffold. (A–C) Confocal images comparing the subcellular localization of the wild-type BcPI-PLC (A) with the H32A (B) and H82A (C) mutants in HEK293-AT1 cells (scale bars, 10 µm). (D and E) Enlarged views of the active sites from the myo-inositol–bound structures of BcPI-PLC (PDB accession no. 1PTG) and BcPI-PLC^H82A (E; PDB accession no. 6S2A). The amino acid side chains coordinating the inositol headgroup are shown as stick representations, and the contacts made between these residues and the myo-inositol headgroup are shown as dashed yellow lines. (F) Structural comparison and threaded alignment (right) of the BcPI-PLC (gold, left) and BcPI-PLC^H82A mutant (blue, center) bound to myo-inositol.

Figure S2.

Tuning the catalytic activity of the FKBP–BcPI-PLC^AA scaffold. (A and B) Published values of the relative in vitro catalytic activities of Bacillus PI-PLC mutants with residues altered within either the enzyme active site (A; adapted with permission from Gässler et al., 1997 and Hondal et al., 1998) or at the membrane-binding interface (B; adapted with permission from Feng et al., 2002, 2003). Please note that the values presented in B for the interfacial mutants were measured using the closely related Bacillus thuringiensis PI-PLC (UniProt ID: P08954). (C) List of the FKBP–BcPI-PLCs variants tested as part of the catalytic activity screen carried out using the mito-DAG^BRET reporter. Briefly, mutagenesis of two interfacial tryptophan residues (W47A/W242A) render the resulting FKBP–BcPI-PLC^AA enzyme entirely cytosolic, thereby significantly reducing the ability of the enzyme to hydrolyze membrane-embedded PI. To further tune the catalytic activity of the parent FKBP–BcPI-PLC^AA scaffold, we screened active site mutants containing alanine or conservative substitutions of R69 (R69A, red traces), R163 (R163A, blue traces; R163K, magenta traces), or Y200 (Y200A, gold traces; Y200F, orange traces). These mutants were tested for their ability to generate DAG in the OMM using the compartment-selective mito-DAG^BRET biosensor. As internal controls, we used the most active parent FKBP–BcPI-PLC^AA scaffold (green traces) and the catalytically inactive H32A variant (gray traces). (D) The raw BRET ratios are shown for triplicate measurements treated with DMSO. Note the differing basal BRET ratios, which reflect the mitochondrial DAG content, as a readout of the background activity of the enzymes from the cytosol. (E) Rapamycin-induced recruitment of the BcPI-PLC variants to the mitochondria results in the rapid production of DAG, which reflects the relative activity of the associated enzyme active site. (F) Normalized BRET ratios are shown after dividing the rapamycin-treated wells by their time-matched DMSO controls. (G) Alternatively, the vehicle-normalized traces from each of the active enzymes can also be presented relative to the kinetics of the inactive FKBP–BcPI-PLC^AA H32A control. After comparing the baseline values, as well as the rapid initial rises in DAG production after rapamycin treatment, we selected the FKBP–BcPI-PLC^AA R163A, R163K, and Y200F mutants as the best of the available variants. These constructs possess a combination of low background activity before recruitment as well as robust catalysis after recruitment onto the membrane surface and were chosen for further exmaination in single-cell imaging studies.

Figure 2.

Steady-state localization of the BcPI-PLC^H82A probe. (A) Comparison of the subcellular localization of the EGFP–BcPI-PLC^H82A probe in COS-7 (left), HEK293-AT1 (center), and HT-1080 (right) cells (scale bars, 10 µm). (B–D) Confocal images of COS-7 cells coexpressing EGFP–BcPI-PLC^H82A with the indicated organelle-specific markers or stably loaded with MitoTracker Red (D; Airyscan detector), which partitions into the mitochondrial matrix (scale bars, 5 µm). Enlarged views of the regions identified by the arrowheads are provided on the far right of each image series (inset, 7.5 µm).

Figure S3.

In vitro binding studies using the BcPI-PLC^ANH mutant. (A) Sequence alignment of the αG helix region of BcPI-PLC (UniProt ID: P14262) and SaPI-PLC (UniProt ID: P45723) shows that two aromatic residues in the BcPI-PLC, Y247 and Y251, which are involved in forming the PC-coordinating cation-π box are replaced by asparagine (N254) and histidine (H258) residues, respectively, in the SaPI-PLC. As described in the Results section, we reasoned that changing Y247 and Y251 to the corresponding residues from SaPI-PLC could diminish the PC sensitivity of the BcPI-PLC scaffold to reveal the PI-dependent binding of the BcPI-PLC^H82A mutant. For simplicity, the resulting BcPI-PLC H82A/Y247N/Y251H mutant is referred to as BcPI-PLC^ANH. (B) Ribbon representation of the BcPI-PLC (blue; PDB accession no. 1PTG) bound to myo-inositol is shown with the αG helix colored in gray and the side chains of residues Y247 and Y251 highlighted in yellow. Please note that these residues are situated on the outside of the molecule, away from the active site, and are positioned immediately below the W242 residue (red) that is also essential for interfacial binding. (C) The in vitro binding of recombinant GFP–BcPI-PLC^ANH to LUVs with varying DAG compositions (0%, 5%, and 10%) was measured using FCCS both in the absence (black bars) and presence (blue bars) of 10% liver PI. Binding measurements are presented as mean values ± SEM from three independent experiments.

Figure S4.

Steady-state localization of the BcPI-PLC^ANH probe. (A–D) Representative images of HEK293-AT1 cells coexpressing EGFP–BcPI-PLC^ANH with an integral Golgi-localized protein fragment (A; FRB-mCherry-Giantin_tail; scale bar, 10 µm), the C-terminal localization signal from the ER-resident protein Sac1 (B; mRFP-Sac1_521-587; scale bar, 10 µm), MitoTracker Red (C; scale bar, 5 µm), or a fluorescently tagged consensus peroxisomal-targeting sequence (D; mRFP-SKL; scale bar, 10 µm). Enlarged views of the regions identified by the arrowheads are provided on the far right of each image series. Note that the size of the inset roughly matches the scale bar that is included for the corresponding organelle marker.

Figure S5.

Mutagenesis of the BcPI-PLC membrane-binding interface limits cytosolic activity. (A) An enlarged view of the membrane-oriented BcPI-PLC interface (top row, left) with the hydrophobic residues W47 and W242 highlighted (yellow sticks; PDB accession no. 1PTG). Images of HEK293-AT1 cells expressing the wild type or indicated mutants of EGFP–BcPI-PLC (scale bars, 10 μm). (B–D) FKBP-tagging of the cytosolic BcPI-PLC^AA was done for acute recruitment to FRB-tagged membranes. Coexpression of the high-affinity DAG-binding probe (mRFP-PKDC1_ab), which labels the direct hydrolytic product of BcPI-PLC activity, should reveal any residual activity of the modified enzymes from the cytosol. Representative images of the DAG-binding probe (bottom panels) coexpressed together with the parent mRFP-FKBP–BcPI-PLC^AA scaffold or its mutated variants are shown (top panels; scale bars, 10 µm).

Figure 3.

Acute manipulation of PI content in membranes of the Golgi complex and ER. (A) Schematic depicting rapamycin-induced recruitment of the FKBP–BcPI-PLC^AA scaffold onto FRB-labeled membrane compartments to locally hydrolyze PI and generate DAG. (B and C) Representative images showing recruitment of the catalytically active mRFP-FKBP–BcPI-PLC^AA R163A (left side panels) or inactive mRFP-FKBP–BcPI-PLC^AA H32A (C, left side panels) enzymes to the surface of the Golgi complex (scale bars, 10 µm) upon 5-min treatment with rapamycin (100 nM). Images on the right show the corresponding changes in the subcellular distribution of the DAG-binding probe (GFP-PKDC1_ab) at 5 and 20 min following rapamycin-dependent enzyme recruitment. (D and E) For each BRET measurement, a schematic of the experimental design is provided above each quantified trace, with the question mark indicating the lipid being measured. Kinetics of DAG production at the Golgi complex (D) or ER (E) after recruitment of the FKBP–BcPI-PLC^AA R163A to the corresponding membrane compartment, as measured using the Golgi-DAG^BRET and ER DAG^BRET biosensors, respectively. A time-matched but alternatively scaled trace shows the compartment-specific FRB:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6, A and B [FRB-Golgi and FRB-ER, respectively]). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure S6.

Kinetics of compartment-specific recruitment of the FKBP–BcPI-PLC^AA scaffold. (A–G) Representative images of HEK293-AT1 cells are shown along with population-level BRET measurements defining the FRB:FKBP dimerization kinetics for the FKBP–BcPI-PLC^AA H32A scaffold upon rapamycin-induced recruitment to the Golgi complex (A), ER (B), OMM (C), peroxisomes (D), PM (E), Rab5-positive compartments (F), or Rab7-positive compartments (G). For each image series, the localization of the FRB-tagged recruiter is shown (left panels) followed by images of the mRFP-FKBP–BcPI-PLC^AA H32A protein before (center panels) and 5 min after (right panels) treatment with rapamycin (100 nM; scale bars, 10 µm). For each of these BRET measurements, the single-plasmid design of the biosensors was adapted such that sLuc was tagged with FKBP–BcPI-PLC^AA H32A and the mVenus fluorescent protein was conjugated to FRB and the respective membrane-specific targeting sequence. For further details related to the design of these constructs, please refer to the Materials and methods section. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells. (H) Summary table listing the most relevant FKBP–BcPI-PLC^AA variants and FRB-tagged organelle recruitment constructs for use in either imaging studies or BRET experiments.

Figure 4.

PI is enriched in the OMM. (A–H) For each BRET measurement, a schematic of the experimental design is provided to the left side of each quantified trace, with the question mark indicating the lipid being measured. (A and B) Kinetics of DAG production within the OMM following recruitment of FKBP–BcPI-PLC^AA R163A to the mitochondria, as measured using the mito-DAG^BRET biosensor. (C) Representative images of HEK293-AT1 cells showing the mitochondrial recruiter (mito-FRB, left) and the localization of the DAG-binding probe (EGFP-PKDC1_ab) before and 5 min after rapamycin-induced recruitment of FKBP–BcPI-PLC^AA R163A to the OMM (scale bar, 10 μm). (D and E) Kinetics of PI4P production within the OMM after recruitment of FKBP-PI4KA^ΔN to the mitochondria, as measured by the mito-PI4P^BRET biosensor. (F) Representative images of cells showing the mitochondrial recruiter (mito-FRB, left) as well as the localization of the PI4P-binding probe (EGFP-P4M_SidM) before and 10 min after rapamycin-induced recruitment of FKBP-PI4KA^ΔN to the OMM (scale bar, 10 μm). (G and H) Kinetics of BcPI-PLC^H82A localization to the OMM after recruitment of either the parent FKBP–BcPI-PLC^AA scaffold (magenta trace) or the modified FKBP–BcPI-PLC^AA R163A (green trace) variant to the mitochondria, as measured using the mito-H82A^BRET biosensor. (I) Representative images of cells showing the mitochondrial recruiter (mito-FRB, left) as well as the localization of the EGFP–BcPI-PLC^H82A probe before and 10 min after rapamycin-induced recruitment of FKBP–BcPI-PLC^AA R163A to the OMM. In B and H, time-matched but alternatively scaled traces show the mito-FRB:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 C). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells (scale bar, 5 μm).

Figure S7.

BRET measurements using the BcPI-PLC^ANH probe. (A) Kinetics of BcPI-PLC^ANH localization to the OMM, as measured using the mito-ANH^BRET biosensor, after mitochondrial recruitment of FKBP–BcPI-PLC^AA R163A. (B) Kinetics of BcPI-PLC^ANH localization to the cytosolic leaflet of the PM, measured using the PM-ANH^BRET biosensor, after PM recruitment of FKBP-Pseudojanin. (C) Kinetics of BcPI-PLC^ANH localization to the cytosolic leaflet of the PM, measured using the PM-ANH^BRET biosensor, in response to treatments with 10 nM (blue trace), 30 nM (magenta trace), or 100 nM (green trace) of the PI4KA-selective inhibitor GSK-A1. (D) Kinetics of BcPI-PLC^ANH levels within the PM, measured using the PM-ANH^BRET biosensor, after treatment with AngII (100 nM). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure 5.

PI is enriched in the cytosolic leaflet of peroxisomes. (A) Representative images of COS-7 cells coexpressing EGFP–BcPI-PLC^H82A and a luminally targeted peroxisomal marker (mRFP-SKL) obtained using either conventional confocal microscopy (top row; scale bar, 10 µm) or with the Airyscan detector (bottom row; scale bar, 2.5 µm). Enlarged views of the regions identified by the arrowheads are provided on the far right of each image series (inset, 10 and 2.5 µm, respectively). (B and C) For each BRET measurement, a schematic of the experimental design is provided above each quantified trace, with the question mark indicating the membrane lipid being measured. (B) Kinetics of DAG production in the cytosolic leaflet of peroxisomes after recruitment of FKBP–BcPI-PLC^AA R163A, as measured by the PEX-DAG^BRET biosensor. A time-matched but alternatively scaled trace shows the PEX-FRB:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 D). (C) Kinetics of PI4P production within the cytosolic leaflet of peroxisomes following recruitment of FKBP-PI4KA^ΔN, as measured by the PEX-PI4P^BRET biosensor. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure 6.

Steady-state levels of PI are low in the PM. (A) Confocal images of HEK293-AT1 cells coexpressing EGFP–BcPI-PLC^H82A with the PI4P-binding probe, mCherry-P4M_SidM (scale bar, 10 µm). An enlarged view of the region identified by the arrowhead is shown on the far right (inset, 10 µm). (B and C) For each BRET measurement, a schematic of the experimental design is provided above each quantified trace, with the question mark indicating the membrane lipid being measured. (B) Kinetics of DAG production within the cytosolic leaflet of the PM, measured by the PM-DAG^BRET biosensor, after recruitment of FKBP–BcPI-PLC^AA R163A to the PM. Please note that a time-matched but alternatively scaled trace shows the PM-FRB:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 E). (C) Kinetics of DAG production within the PM, measured using the PM-DAG^BRET biosensor, in response to stimulation with AngII (100 nM; gray trace) or following PM recruitment of an FKBP-tagged mammalian PLC (FKBP-PLCδ1^Δ44,ΔPH; orange trace). For comparison, the normalized DAG response measured after the recruitment of FKBP–BcPI-PLC^AA R163A, which is presented in B, is also included (yellow trace). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells. (D) Representative images from cells coexpressing the DAG-binding probe (mRFP-PKDC1_ab; left panels) and EGFP–BcPI-PLC^H82A (right panels; scale bars, 10 µm). Note the massive translocation of the DAG probe, but not the EGFP–BcPI-PLC^H82A, from the cytosol to the PM after stimulation with AngII (100 nM). The enlarged image of the area marked by the red inset in the center panels is presented on the right of each image series (15-µm inset).

Figure S8.

PI availability within the PM is controlled by delivery from the ER and local conversion to PPIn species. (A–D) For each BRET measurement, a schematic of the experimental design is provided to the left side of each quantified trace, with the question mark indicating the membrane lipid being measured. (A) Kinetics of PI4P production within the cytosolic leaflet of the PM after recruitment of FKBP-PI4KA^ΔN to the PM, as measured using the PM-PI4P^BRET biosensor. (B) Kinetics of BcPI-PLC^H82A localization to the cytosolic leaflet of the PM, measured using the PM-H82A^BRET biosensor, after PM recruitment of the tandem PPIn phosphatase, Pseudojanin, or its inactive mutant. (C) Kinetics of BcPI-PLC^H82A localization to the cytosolic leaflet of the PM in response to treatments with 10 nM (blue trace), 30 nM (magenta trace), or 100 nM (green trace) of the PI4KA-selective inhibitor GSK-A1, as measured using the PM-H82A^BRET biosensor. (D) Kinetics of DAG production within the cytosolic leaflet of the PM, measured using the PM-DAG^BRET biosensor, from cells pretreated with DMSO (yellow trace) or GSK-A1 (100 nM; magenta trace) for 30 min before recruitment of the FKBP–BcPI-PLC^AA R163A enzyme to the PM. Please note that a time-matched but alternatively scaled trace shows the PM-FRB:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 E). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure 7.

PPIn production within the PM depends on PI delivery from the ER. (A–C and E–G) A schematic depicting the experimental design is provided above confocal images of representative cells showing the recruitment of FKBP–BcPI-PLC^AA R163A to the PM (B and F, left panels, green) or to the ER (C and G, left panels, magenta) after a 5-min treatment with rapamycin (100 nM). The localization of PI4P (B and C; EGFP-P4M_SidMx2) and PI(4,5)P₂ (F and G; PLCδ1_PH-EGFP) are provided before (center panels) and 20 min after (right panels) recruitment of FKBP–BcPI-PLC^AA R163A to the respective membrane compartments (scale bars, 10 μm). (D and H) Kinetics of PI4P (D) or PI(4,5)P₂ (H) levels within the cytosolic leaflet of the PM as measured using the PM-PI4P^BRET or PM-PI(4,5)P₂^BRET biosensors, respectively, in response to recruitment of FKBP–BcPI-PLC^AA R163A either directly to the PM (PM-FRB; green traces) or to the ER (FRB-ER; magenta traces). Treatment with GSK-A1 (100 nM; D, grey trace), which selectively inhibits PI4KA, or AngII (100 nM; H, grey trace), which stimulates PI(4,5)P₂ hydrolysis, are included as positive controls for the PM-PI4P^BRET and PM-PI(4,5)P₂^BRET biosensors, respectively, as well as to provide scale for any changes to PPIn levels that are associated with the membrane recruitment of FKBP–BcPI-PLC^AA R163A. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure S9.

PM and ER localization of the BcPI-PLC^H82A probe in response to endogenous PLC activation. (A–C) A summary schematic of the experimental design is presented alongside the kinetics of BcPI-PLC^H82A (B and C; PM-H82A^BRET), PI4P (C; PM-PI4P^BRET), and PI(4,5)P₂ (C; PM-PI(4,5)P₂^BRET) levels within the PM after treatment with AngII (100 nM). Please note that the green BRET trace shown in B has been expanded in C to scale the magnitude of the changes observed. (D) In addition to the PM, the levels of BcPI-PLC^H82A (ER-H82A^BRET) within the ER are also shown after AngII stimulation (100 nM). BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure 8.

PI4P levels at the Golgi complex are sensitive to the local availability of PI. (A) Confocal images of COS-7 cells coexpressing EGFP–BcPI-PLC^H82A together with the PH domain of FAPP1 (FAPP1_PH-EGFP; scale bar, 5 µm). An enlarged view of the region identified by the arrowhead is shown on the far right (inset, 10 µm). (B, C, E, and F) Schematics depicting the experimental design are provided above images of representative HEK293-AT1 cells showing the recruitment of FKBP–BcPI-PLC^AA R163A (C and F; top row, left panels, green) or FKBP–BcPI-PLC^AA (C and F; bottom row, left panels, magenta) to the Golgi complex (C; FRB-Golgi) or ER (F; FRB-ER) after a 5-min treatment with rapamycin (100 nM). Localization of the FAPP1_PH-EGFP probe is shown before and 20 min after initiation of localized PI hydrolysis by recruitment of the BcPI-PLC mutants to the Golgi (C; FRB-Golgi) or ER (F; FRB-ER). Scale bars in C and F are 10 μm. (D and G) Pooled image analyses measuring changes in FAPP1_PH-EGFP intensities at the perinuclear Golgi region after recruitment of FKBP–BcPI-PLC^AA R163A (green traces; D, 46 cells; G, 42 cells), mRFP-FKBP–BcPI-PLC^AA (magenta traces; D, 16 cells; G, 21 cells), or mRFP-FKBP–BcPI-PLC^AA H32A (gray traces; D, 43 cells; G, 42 cells) to the respective compartments (D, Golgi; G, ER). Normalized intensities (F_(t)/F_pre) of the FAPP1_PH-EGFP signal at the perinuclear Golgi region, relative to the cytosolic fraction, are presented as mean values ± SEM from a minimum of four independent experiments. The pretreatment period used for normalization was defined as the average ratio of the Golgi/cytosolic signal intensity measured over the first four frames of each recording.

Figure 9.

Maintenance of PI3P levels in Rab5-positive endosomes requires the delivery of PI from the ER. (A) Confocal images of HEK293-AT1 coexpressing the EGFP–BcPI-PLC^H82A probe together with mCherry-Rab5^WT (scale bars, 5 µm). An enlarged view of the region identified by the arrowhead is shown on the far right (inset, 2.5 µm). (B and C) For each BRET measurement, a schematic of the experimental design is provided above each quantified trace, with the question mark indicating the membrane lipid being measured. (B) Kinetics of DAG production in Rab5-positive compartments after recruitment of FKBP–BcPI-PLC^AA R163A to Rab5-labeled membranes, as measured by the Rab5-DAG^BRET biosensor. Please note that a time-matched but alternatively scaled trace shows the FRB-Rab5:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 F). (C) Kinetics of PI3P levels within Rab5-positive compartments, measured using the biosensor (C; Rab5-PI3P^BRET), in response to the recruitment of FKBP–BcPI-PLC^AA R163A either directly to the surface of Rab5-labeled endosomes (green traces; FRB-Rab5) or to the ER (magenta traces; FRB-ER). Treatment with a selective class III PI 3-kinase inhibitor (VPS34-IN1, 300 nM; gray traces) is included as a positive control for the Rab5-specific PI3P^BRET biosensor and to provide scale for any changes associated with the differential recruitment of FKBP–BcPI-PLC^AA R163A to Rab5-positive endosomes or the ER. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.

Figure 10.

Maintenance of PI3P levels in Rab7-positive endosomes requires the delivery of PI from the ER. (A) Confocal images of HEK293-AT1 coexpressing the EGFP–BcPI-PLC^H82A probe together with mCherry-Rab7^WT (scale bars, 5 µm). An enlarged view of the region identified by the arrowhead is shown on the far right (inset, 2.5 µm). (B and C) For each BRET measurement, a schematic of the experimental design is provided above each quantified trace, with the question mark indicating the membrane lipid being measured. (B) Kinetics of DAG production in Rab7-positive compartments after recruitment of FKBP–BcPI-PLC^AA R163A to Rab7-labeled membranes, as measured by the Rab7-DAG^BRET biosensor. Please note that a time-matched but alternatively scaled trace shows the FRB-Rab7:FKBP–BcPI-PLC^AA H32A dimerization kinetics (red line; see also Fig. S6 G). (C) Kinetics of PI3P levels within Rab7-positive compartments, measured using the Rab7-PI3P^BRET biosensor, in response to the recruitment of FKBP–BcPI-PLC^AA R163A either directly to the surface of Rab7-labeled endosomes (green traces; FRB-Rab7) or to the ER (magenta traces; FRB-ER). Treatment with a selective class III PI 3-kinase inhibitor (VPS34-IN1, 300 nM; gray traces) is included as a positive control for the Rab7-specific PI3P^BRET biosensor and to provide scale for any changes associated with the differential recruitment of FKBP–BcPI-PLC^AA R163A to Rab7-positive endosomes or the ER. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.