Protein expression and purification
The gene encoding LYCHOS (GPR155) (Uniprot Q7Z3F1), was synthesized and subcloned into pTwist CMV BG WPRE Neo (Twist Bioscience) with a C-terminal Flag tag for mammalian expression. For insect cell expression, LYCHOS (Uniprot Q7Z3F1) was synthesized and cloned (Genscript) into pFastBac1 (Thermo Fisher Scientific) with a C-terminal Flag tag. All mutants were generated by site-directed mutagenesis (GenScript or in-house) and sequences verified by whole-plasmid sequencing. Throughout our investigation, we routinely compared mammalian-derived and insect-derived recombinant LYCHOS in an attempt to capture different protein conformations using cryo-EM. However, our assessments with cryo-EM, surface plasmon resonance, mass photometry and size-exclusion chromatography revealed no significant differences in LYCHOS behaviour between the two expression systems. SLC10A1 was synthesized and cloned (GenScript) into pFASTBac1 (Thermo Fisher Scientific) with a C-terminal Twin-Strep-tag for insect cell expression.
For mammalian expression, LYCHOS was expressed in Expi293F cells (Thermo Fisher Scientific) and passaged in Expi293 Expression Medium (Thermo Fisher Scientific). Cells were transfected with polyethylenimine (PEI) Max (Polysciences) at a PEI:DNA ratio of 5:1, and a final DNA amount of 1.25 μg ml−1. Recombinant proteins were expressed for 72 h at 37 °C, and cells were supplemented with 5% CO2. Cells were collected by centrifugation and frozen at –80 °C before purification. For insect cell expression, LYCHOS or indicated mutants were expressed in Sf9 cells for three days after infection of each 1-l culture with 4 ml of baculovirus, produced by following the manufacturer’s protocol (Bac-to-Bac, Invitrogen). Cells were collected by centrifugation, washed in phosphate-buffered saline (PBS) and stored at −80 °C until use.
Cells were resuspended in buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1% (w/v) lauryl maltose neopentyl glycol (LMNG), 0.1% (w/v) CHS and one EDTA-free protease inhibitor cocktail tablet (Roche) per litre of cell culture. The cells were incubated at 4 °C for 3 h with stirring to solubilize the membranes. Subsequently, the solubilized material was centrifuged at 20,000 rpm for 1 h, followed by filtration of the supernatant through a 0.8 µm filter. Anti-Flag G1 affinity resin (GenScript) was added and incubated for 30 min at 4 °C on a horizontal roller. Flag resin was collected by centrifugation, washed and transferred to a gravity flow column (BioRad). The resin was washed with 20 column volumes (CVs) of 0.1% (w/v) LMNG and 0.01% (w/v) CHS buffer, followed by 20 CVs of 0.05% (w/v) LMNG and 0.005% (w/v) CHS buffer. Protein was eluted with 0.1 mg ml−1 Flag peptide (Genscript) in 0.002% (w/v) LMNG and 0.0002% (w/v) CHS buffer. Fractions containing protein were concentrated and further purified by size-exclusion chromatography using a Superose 6 10/300 column (Cytiva) equilibrated with 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT, 0.002% (w/v) LMNG and 0.0002% (w/v) CHS. Peak fractions were concentrated by a factor of 40−50 to approximately 20 µM using a centrifugal concentrator with a 100-kDa molecular weight cut-off and snap-frozen before storage at −80 °C. SLC10A1–STREP was expressed in Sf9 cells and purified as described for LYCHOS except that the Flag-affinity step was substituted for Strep-tag purification using a 1-ml StrepTrap XT prepacked column (Cytiva) and the protein was eluted with 50 mM biotin.
Surface plasmon resonance
Surface plasmon resonance experiments were performed using Sf9 cell expressed recombinant LYCHOS on a Biacore T200 (GE Healthcare) in running buffer containing 10 mM HEPES (pH 7.4), 300 mM NaCl, 0.005% (w/v) LMNG, 0.0005% CHS (w/v), 0.005% NP-40 (v/v) and 2% (v/v) dimethyl sulfoxide (DMSO) at 25 °C with a flow rate of 30 µl min−1. To measure the binding of IAA (175.18 g mol−1) and tryptophan (204.23 g mol−1) to LYCHOS, we generated high-density LYCHOS immobilized on a CM7 sensor chip (GE Healthcare). In brief, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) standard amine coupling was performed by passing LYCHOS at 200 µg ml−1 (1 µM, 193.8 kDa) in sodium acetate buffer (100 mM, pH 4.5) at 10 µl min−1. Immobilization levels of 25,000–30,000 response units (RU) were reached to measure the small-molecule ligands (theoretical RMax ≈ 36–54, respectively, assuming two binding sites). Here, SLC10A1, a known sodium/taurocholate co-transporter with a similar protein fold was chosen as the reference to improve subtraction of baseline and non-specific effects. Immobilized LYCHOS was allowed to stabilize for two to four hours in running buffer at 30 µl min−1 before measurements to ensure a stable baseline. To minimize buffer mismatch effects, stock solutions of tryptophan and IAA were made to 500 mM. The pH was adjusted to 7.4 and twofold serial dilutions of each ligand were made into running buffer. Serial dilutions of IAA and tryptophan (20 mM to 156.3 µM) were injected in ascending order for 60 s and dissociation was monitored for 180 s. Carry-over control injections and needle pre-dipping were performed to prevent sample cross-contamination. Each injection was measured at 10 kHz with a 50% (v/v) DMSO wash after each cycle. Reference and blank subtractions were performed to account for drift, bulk and solvent effects. Affinity, measured from steady-state curves, is expressed as the mean of four to seven experimental replicates. RMax values of 19.8 ± 1.3, 52.4 ± 3.0, 42.8 ± 2.5 and 36.4 ± 2.0 were obtained for immobilized WT (19,544 RU), L177W (30,981 RU), N145A (26,191 RU) and A148W (28,808 RU) variants of LYCHOS, respectively. These values are in line with the expected theoretical RMax, suggesting that the vast majority of immobilized LYCHOS remains in an active state. At least two separate protein preparations were used to conduct affinity measurements, and between five and eight experimental replicates were conducted for each LYCHOS variant. Each experimental replicate consisted of an independent concentration series, which was modelled with a one-to-one binding curve (as defined previously31). Affinity measurements were in excellent agreement between independent protein preparations, sensor chips and IAA solutions.
Cryo-EM sample preparation and data collection
Quantifoil R1.2/1.3 200 mesh copper grids were glow discharged at 30 mA for 30 s using a Pelco easiGlow instrument. Freshly purified LYCHOS (3.5 μl at 10 µM) was immediately applied to grids and rapidly vitrified in liquid ethane. To analyse IAA and tryptophan interactions, solutions of each ligand were made at 100 mM and diluted into the sample to a final concentration of 10 mM. In comparison, an IAA concentration of 15 mM was used to determine the IAA-bound PIN8 structure21. IAA and tryptophan were left to incubate with LYCHOS for 60 s before freezing. In one instance, LYCHOS–IAA was left to incubate for 2 h on ice. The vitrification process was performed with a Vitrobot Mark IV (Thermo Fisher Scientific) after hand blotting with Fisherman Grade 1 filter paper. The temperature was maintained at 4 °C with 100% relative humidity. Data were collected on a Titan Krios G1 (Thermo Fisher Scientific) electron microscope operating at 300 kV with a 50 μm C2 aperture. Micrographs were obtained using a Gatan K3 direct electron detector in counting mode at a nominal EF-TEM magnification of 105,000×, corresponding to a calibrated physical pixel size of 0.8234 Å. A Gatan GIF Quantum energy filter was used with a slit width of 10 eV. The electron dose rate was set to 10.7 electrons pixel−1 s−1, with a total exposure time of 3.77 s and a cumulative dose of 60 electrons Å−2 distributed across 60 frames. Automated collection was performed using EPU (v.2.12.1.2782) with beam shift, capturing 21 images per stage movement. The nominal defocus range was set between −0.5 and −2.0 µm.
Multiple wild-type LYCHOS samples were prepared from either Expi293F or Sf9 tissue cultures for electron microscopy. Overall, these samples were indiscernible. We note that during these comparisons, grid variation and ice thickness (for example, when looking at the same preparation of LYCHOS) affected the stability of the DEP domains, which were prone to denaturing. We performed direct side-by-side comparisons of Sf9 wild-type LYCHOS with (PDB 8U5N) and without (PDB 8U54) IAA, both originating from the same protein preparation. Likewise, direct comparisons were performed between Expi293F-produced wild-type LYCHOS (same batch) with tryptophan (PDB 8U58), with IAA (PDB 8U5Q, 8U5V and 8U5X) or without ligand (PDB 8U56). Here, we report two reconstructions of wild-type LYCHOS, which yielded the highest resolution (PDB 8U56; Expi293F) and the most complete sequence coverage (PDB 8U54; Sf9). We collected an additional two datasets after brief incubations with IAA (PDB 8U5Q, 8U5V and 8U5X; Expi293F) and tryptophan (PDB 8U58; Expi293F). Finally, we investigated the W678R/F352A LYCHOS mutation (PDB 8U5C; Sf9) and LYCHOS–IAA after prolonged incubation (PDB 8U5N; Sf9).
Cryo-EM data processing
A total of six datasets were collected with movies ranging from 4,000 to 16,000. Dose-fractionated movies were corrected for beam-induced motion and compensated for radiation damage within MotionCor2 (v.1.1.0)32. Aligned, dose-weighted averages were subsequently imported into cryoSPARC33 for all further processing. The contrast transfer function (CTF) parameters were estimated with CTFFIND (v.4.1.8)34 or by patch-based CTF estimation. Micrographs lacking Thon rings at 5 Å or better were discarded. In the first instance, multiple rounds of autopicking and blob picking in cryoSPARC33 were performed, followed by particle duplicate removal and two-dimensional (2D) classification. Clean classes were used to train a Topaz35 model. This model was used subsequently for all datasets. In the case of monomeric LYCHOS (long incubation with IAA), an extra round of blob picking was performed to ensure no particles were omitted.
All particles were at first extracted in a 400 × 400-pixel box and downsampled by Fourier cropping to 64 corresponding to a pixel size of around 5 Å pixel−1. These were subjected to multiple rounds of 2D classification in both RELION (v.3.1, 4.0b)36 and cryoSPARC33, yielding particles of sufficient quality and homogeneity for three-dimensional (3D) classification. Typically, this was performed once only to remove clear false positives and contamination. These were re-extracted, centred and downsampled to 128 × 128, for a pixel size of around 2.5 Å pixel−1. This set was subjected to two rounds of ab initio classification, using four classes (maximum and minimum resolution set to 5 Å and 12 Å, respectively; Fourier radius step, 0.08; initial and final minibatch, 1,500) to filter particles. Typically, a single volume was selected for further refinement. Particles were re-extracted at around 1.1 Å pixel−1 and subjected to non-uniform refinement with C2 symmetry, yielding 3.3 Å maps on average.
Particle polishing was performed in RELION36, re-extracting particles for a final pixel size of 1.108 Å pixel−1, and the particles were re-refined. Here, an additional round of ab initio particle classification was performed. In the case of LYCHOS–IAA (short incubation), two populations were evident in which the GPCR domain seemed to sample from two distinct conformations. Symmetry expansion and focused classification were performed, followed by reconstruction without an additional angular search. Conversions between software were performed with EMAN (v.2.2)37, with code written in-house, or by pyem. The Fourier shell correlation (FSC) was used to estimate resolution at the 0.143 threshold. Local resolution was estimated by the windowed blocres FSC method (0.5 threshold) as implemented in cryoSPARC33. Map sharpening in cryoSPARC33 and EMReady38 was performed to assist in residue assignment and model building.
Model building
In the first instance, a computed model of LYCHOS with C2 symmetry was generated by AlphaFold16 using an A6000 GPU with 48 Gb of VRAM. Domains and appropriate interfaces from this model were split and rigid-body fitted into the EMReady38 sharpened map. Several regions were manually rebuilt and adjusted using a combination of Coot39, ISOLDE40 and ChimeraX41. Finally, real space refinement was conducted in PHENIX (v.1.20.1)42 using harmonic potential restraints. All figures, analysis, video renders and visualizations were produced in ChimeraX41 or with Python and Matplotlib.
SSM-based electrophysiology
SSM electrophysiology was performed on a SURFE2R N1 (Nanion Technologies). Electrode sensors (3 mm) were purchased (Nanion Technologies) and prepared according to established protocols21,43. First, to confirm sensor fidelity, capacitance and conductance measurements were performed in non-activating buffer (20 mM HEPES, pH 8.5, 150 mM NaCl and 1 mM MgCl2). Ideal sensors have capacitance between 60 nF and 80 nF and conductivity between 10 nS and 50 nS. Next, sensors were rinsed with ultrapure water, dried under a gentle N2 (g) stream and treated with 50 µl of 0.5 mM 1-octadecanethiol (Sigma) for 30 min at 25 °C or overnight at 4 °C. To prevent evaporation, an inverted Petri dish with a small volume of distilled water was used to form an airtight seal. Thiol solution was removed by inverting the sensor and gently tapping the sensor to filter paper, followed by generous rinsing with 100% (v/v) isopropanol and ultrapure water. The sensor was again dried under a gentle N2 (g) stream. To prepare the SSM, a 2.0 µl droplet of a 7.5 mg ml−1 solution of 1,2-diphytanoylphosphatidylcholine (Avanti) dissolved in n-decane was applied directly to the clean and dry gold surface using a Hamilton pipette, ensuring a uniform coverage of the 3 mm sensor surface. Immediately, 50 µl of non-activating buffer was applied and the SSM was left to assemble at 25 °C for 1 h in an airtight chamber. Sensors were again validated for capacitance and conductance.
To prepare LYCHOS embedded vesicles, 10 mg of soy phospholipid mixture (38% w/v phosphatidylcholine, 30% w/v phosphatidyl ethanolamine, 18% w/v phosphatidyl inositol, 7% w/v phosphatidic acid and 7% w/v other soy lipids; Avanti) was dispended into glass vials and dried under argon (g). Lipid films were then resuspended in activating buffer to 3 mg ml−1 and extruded through a polycarbonate filter (ø 200 nm) to form unilamellar vesicles. Proteoliposomes containing LYCHOS were assembled by the LAiR method44. Here, 5 µg wild-type LYCHOS (Sf9; 3.6 mg ml−1, 17 µM) was diluted into 10 µl liposomes and allowed to incubate for 15 min, before further dilution with 40 µl non-activating buffer. Next, 50 µl proteoliposomes was applied to prepared SSMs and sensors were centrifuged at 4,000g for 30 min at 25 °C in a custom 3D-printed apparatus. Sensors were subsequently washed with non-activating buffer.
Typical single solution exchange experiments were conducted. In brief, a single measurement at a given concentration consisted of a 1-s pulse of non-activating buffer at 200 µl s−1 to establish a baseline, immediate (<30 ms) injection of a 1-s pulse of activating buffer to induce transient currents, and a 1-s pulse of non-activating buffer to exchange the system back to a resting state (total of 3 s). After each measurement, a 2-s pulse with non-activating buffer was performed to equilibrate the sensor. Current transients were recorded for a full concentration series of IAA (0–30 mM). For each concentration series, a corresponding blank sensor measurement series was performed. Measurements were performed on at least two individual sensors. Each concentration series was conducted independently for a total of four experimental replicates (as described previously21). Finally, to describe the peak currents in response to IAA, we fitted a Michaelis–Menten curve to the peak currents, after blank subtraction and normalization (I/IMax, to account for different quantities of LYCHOS across sensors).
Mass photometry
To assess the stoichiometry and mass distribution of LYCHOS samples, standard mass photometry landing assays were conducted at 20 °C using the TwoMP instrument (Refeyn) on an active anti-vibration platform. Silicone gaskets and glass coverslips were purchased from Refeyn. Glass coverslips were cleaned by washing in sonication baths of 100% (v/v) isopropanol, ultrapure water and, finally, by plasma glow discharge. To avoid noise originating from LMNG micelles, each sample was maintained in 20 mM HEPES, 150 mM NaCl, 0.002% (w/v) LMNG and 0.0002% (w/v) CHS until measurement. Samples were then diluted 20-fold into detergent-free buffer before immediately measuring a 60–120 s image series. The final protein concentration at the time of measurement was 20 nM. Standards of bovine serum albumin (Merck) and apoferritin (Merck) were measured on the same day to calibrate extracted particle contrast to mass. Image series were acquired with an 8 ms exposure (128 Hz) at 488 nm, with a set field of view of 12 × 17 µm. Frame and pixel binning were applied, with a factor of 3 and 6, respectively, for an effective pixel size of 72 nm. Analysis and acquisition were performed using the Refeyn AquireMP and DiscoverMP packages (v.2.5). To investigate the effect of prolonged incubation of LYCHOS with IAA, samples (Sf9 or Expi293F-derived) were incubated with or without IAA for 2 h at room temperature. Here, we observed no difference between Sf9 or Expi293F-derived protein after incubation with IAA. Time-zero samples were obtained by measuring each reaction immediately before the addition of 10 mM IAA.
Cell culture and cDNAs for transfection
HEK293 cells (ATCC; negative for mycoplasma contamination, not authenticated) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS). The sequence for the IAA FRET biosensor, AuxSen, was a gift from M. Kolb27 and the biosensor was commercially synthesized (Genscript) into pcDNA3.1 (Thermo Fisher Scientific). The pcDNA3-LysoTORCAR construct was a gift from J. Zhang (Addgene plasmid 64929; http://n2t.net/addgene:64929; RRID: Addgene_64929). For LysoTORCAR assays, LYCHOS or LYCHOS mutants were cloned into pcDNA 3.1 (Thermo Fisher Scientific) with a C-terminal Flag tag. For AuxSen efflux assays, LYCHOS was synthesized and cloned into pTwist CMV BG WPRE Neo (Twist Bioscience) with a C-terminal Flag tag. As a positive control for AuxSen assays, the gene encoding PIN8 (Uniprot Q9LFP6), was synthesized and subcloned into pTwist CMV BG WPRE Neo (Twist Bioscience) with an N-terminal Flag tag. LYCHOS-Flag-NLuc and HA-cpmCitrine-NPRL2 constructs were generated in-house and cloned into pcDNA 3.1 (Thermo Fisher Scientific) by Gibson assembly. The genes for cpmCitrine and NLuc were subcloned from the cAMP BRET biosensor, CAMYEN45, a gift from B. Hoare. LYCHOS genes were obtained from pFASTBac1 expression vectors (see ‘Protein expression and purification’) and NPRL2 was subcloned from pRK5 HA-NPRL2, a gift from D. Sabatini and K. Shen (Addgene plasmid 99709; http://n2t.net/addgene:99709; RRID :Addgene_99709). All LYCHOS-NLuc variants were generated in-house using overlap extension PCR, Gibson assembly or QuickChange mutagenesis. The construct for β2-adrenoceptor-NLuc was a gift from C. W. White and S. J. Hill.
AuxSen efflux assay
HEK293 cells were transfected and seeded in suspension in six-well plates using 25 kDa linear PEI at a ratio of 1:6 DNA:PEI. The cells were co-transfected with a FRET biosensor for IAA, AuxSen27 (200 ng per well) and empty vector control, LYCHOS–Flag (WT) or Flag–PIN8 at 1 μg per well, to mislocalize LYCHOS and PIN8 to the plasma membrane (Extended Data Fig. 7k; as described previously19,21). After 24 h, cells were reseeded into a poly-d-lysine-coated black, optically clear 96-well plate (Perkin Elmer ViewPlate), and left to adhere for 24 h. On the day of the experiment, medium was removed and cells were equilibrated in Hank’s balanced salt solution (HBSS; Invitrogen) at room temperature. Fluorescence imaging was performed using a high-content Perkin Elmer Operetta with an Olympus LUCPlanFLN 20× NA 0.45 objective and Harmony software (v.4.8) as previously described46, with some modifications. For emission ratio analysis, cells were excited sequentially (410–430 nm excitation filter) with emission measured using 520–560 nm and 460–500 nm emission filters. Cells were imaged every 1 min. Before imaging, 25 μM NPA was added to PIN8-transfected cells, as stated. Baseline emission ratio images were captured for 5 min before the addition of 10 μM IAA, and influx emission ratio images were captured for 15 min (to allow IAA influx to reach a stable plateau). IAA was then removed, and cells were placed into HBSS without or with 25 μM NPA (as stated), before efflux ratio images were captured for 25 min. Data were analysed as described previously46 using in-house automated macros within the Fiji distribution of ImageJ (v.2.14.0/1.54f)47. Cells with a greater than 10% change in F/F0 (FRET ratio relative to baseline for each cell) after IAA influx were selected for analysis using Microsoft Excel (v.16.45). Data are expressed as the average emission ratio relative to the total IAA influx (15 min after IAA addition) and efflux (25 min after buffer exchange) for each cell (F/FInflux), and averaged over each biological replicate. To determine the rate of efflux (K), data were fitted using a plateau followed by one-phase decay non-linear regression model in GraphPad Prism (v.10.0.3), with X0 constrained to 0, Y0 constrained to 1 and plateau constrained to 0. Data shown were obtained from three independent biological replicates.
LysoTORCAR mTORC1 activity assay
HEK293 cells were co-transfected in six-well plates with a lysosomally localized FRET biosensor for mTORC1, LysoTORCAR29 (500 ng per well), and empty vector control, LYCHOS–Flag (WT), LYCHOS–Flag (Y57A), LYCHOS–Flag (F352A), LYCHOS–Flag (F352A/W678R), LYCHOS–Flag (N145A) or LYCHOS–Flag (F362A/W678R/N145A) (500 ng per well), using X-tremeGENE at a 1:3 DNA:reagent ratio. After 24 h, cells were replated into a black, optically clear 96-well plate (Perkin Elmer ViewPlate), pre-coated with poly-d-lysine. After six hours, cells were serum starved overnight in DMEM (no FBS). On the day of the experiment, cells were amino acid starved for 1 h in HBSS at 37 °C before fluorescence imaging using a high-content Perkin Elmer Operetta with an Olympus LUCPlanFLN 20× NA 0.45 objective and Harmony software (v.4.8). Cells were excited sequentially (410–430 nm excitation filter) with emission measured using 520–560 nm and 460–500 nm emission filters. Cells were imaged every one minute. Baseline emission ratio images were captured for 5 min before complete buffer exchange to phenol red-free DMEM alone (control), or containing 1.3% (w/v) methyl-β-cyclodextrin (MβCD) (cholesterol depletion) or 0.1% (w/v) MβCD/50 μM cholesterol complexes (cholesterol addition)2,48. Emission ratio images were captured for 40 min, before complete buffer exchange for MβCD-treated cells only, to 0.1% (w/v) MβCD/50 μM cholesterol complexes (cholesterol repletion). Emission ratio images were captured for an additional 30 min. Data were analysed as for the AuxSen FRET biosensor using Fiji (v.2.14.0/1.54f) and in-house automated macros. Addition of phenol red-free DMEM caused a change in auto-fluorescence which was corrected using Microsoft Excel (v.16.45). Data are expressed as the average change in FRET ratio relative to baseline for each cell (F/F0), and averaged over each biological replicate. The AUC and the fold change induced by a stimulus (average of five time points at peak response relative to baseline, or relative to the last five time points of MβCD treatment for repletion) were calculated using GraphPad Prism (v.10.0.3). Data shown were obtained from three or four independent biological replicates.
LYCHOS–GATOR1 saturation BRET assay
HEK293 cells were transfected in six-well plates using 25-kDa linear PEI at a ratio of 1:6 DNA:PEI. Cells were co-transfected with 25 ng per well LYCHOS-NLuc WT, N145A, F352A/W678R or F352A/W678R/N145A, or 25 ng per well β2-adrenoceptor-NLuc (negative control), and increasing amounts of cpmCitrine-NPRL2 (0 ng, 135 ng, 270 ng, 405 ng, 810 ng or 1,200 ng). The total amount of DNA was made up to 1,225 ng per well using empty vector. After 24 h, cells were reseeded in quadruplicate into a poly-d-lysine-coated white opaque 96-well plate (Perkin Elmer CulturePlate) at 5 × 104 cells per well. After another 24 h, cells were washed and equilibrated with assay buffer (10 mM HEPES in HBSS, pH 7.4 at 37 °C) for 25 min. Cells were then incubated for 5 min at 37 °C with the NanoGlo substrate (1:1,000; Promega N1120). BRET measurements were obtained with the LUMIstar microplate reader (BMG Labtech; Omega control software v.6.20), which enabled simultaneous measurement of luminescence (NLuc; 445–505 nm) and cpmCitrine emission (505–565 nm) in individual wells. At the end of the experiment, total cpmCitrine fluorescence was measured with an excitation of 482–512 nm, dichroic filter set at 517.2 nm and emission at 520–560 nm using the CLARIOstar microplate reader (BMG Labtech, SMART Control software v.4.20) to determine the relative amount of cpmCitrine-NPRL2 expressed in cells. BRET ratios were calculated as follows: cpmCitrine emission (505–565 nm)/NLuc emission (445–505 nm). Data are expressed as the net BRET ratio relative to the ratio of cpmCitrine/NLuc emission for each transfection condition (relative expression of each protein in the cells), and were fitted using a non-linear regression one-site-specific binding model in GraphPad Prism (v.10.0.3) to obtain BRET50 (Kd) and BRETMax (Bmax) values. For comparisons between LYCHOS mutants, data are expressed as the net BRET relative to the BRETMax for each condition, determined from the non-linear regression one-site-specific binding model. Data are from three to five independent biological replicates.
Western blot expression analysis
To confirm equivalent expression of LYCHOS–Flag WT or mutants for the LysoTORCAR experiments, HEK293 cells were transfected with the same amount of DNA in six-well plates (500 ng per well LYCHOS–Flag WT, Y57A, F352A or N145A) using X-tremeGENE (as described above). After 48 h, cells were collected in ice-cold PBS, and pellets were resuspended in ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% v/v Triton X-100, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS, protease inhibitor cocktail, phosphatase inhibitor cocktail, 1 mM benzamidine and 1 mM PMSF). Cells were incubated on ice with agitation for 20 min, then lysates were centrifuged at 14,000g for 10 min at 4 °C, and supernatants were recovered. Total protein concentration was quantified using a Pierce BCA Protein Assay Kit, and 35 μg was run on 4–15% Mini-PROTEAN pre-cast gels (BioRad). Proteins were transferred to nitrocellulose membranes by electroblotting using the Trans-Blot Turbo Transfer System (BioRad) and the STANDARD SD transfer protocol. The membranes were blocked with 5% (w/v) BSA, 0.02% (w/v) sodium azide in Tris-HCl-buffered saline (TBS) with 0.1% (v/v) Tween-20 (TBS-T) for 1 h. The blots were incubated overnight at 4 °C with mouse anti-Flag (Merck, F3165; 1:5,000), or rabbit anti-β-tubulin (Cell Signaling Technology 2146; 1:1,000) in 5% (w/v) BSA, 0.02% (w/v) sodium azide in TBS-T. Blots were washed three times with TBS-T before incubation with IRDye 680RD goat anti-mouse and IRDye 800CW goat anti-rabbit fluorescent secondary antibodies (LICOR 926-68070 and 926-32211) at a 1:15,000 dilution in 5% (w/v) BSA, 0.02% (w/v) sodium azide in TBS-T for 1 h. Blots were then washed three times in TBS-T and once in TBS, and bands were visualized with an Amersham Typhoon 5 (control software v.3.0.0.2). Densitometry of bands was determined with Fiji (v.2.14.0/1.54f). Data are expressed relative to the loading control (β-tubulin) and are from three independent biological replicates.
Confocal imaging for plasma-membrane localization of Flag-tagged LYCHOS and PIN8
To confirm that some LYCHOS–Flag and Flag–PIN8 were mislocalized to the plasma membrane for the AuxSen experiments (and not under normal transfection conditions), HEK293 cells were transfected and seeded in six-well plates with LYCHOS–Flag (WT) or Flag–PIN8 (both 500 ng per well or 1 μg per well) in suspension using 25-kDa linear PEI at a ratio of 1:6 DNA:PEI. After 24 h, cells were reseeded into a black optically clear 96-well plate (Perkin Elmer PhenoPlate). After 24 h, cells were washed three times in ice-cold PBS, before fixation in 4% (v/v) paraformaldehyde for 15 min at room temperature. After three washes with PBS (5 min each), cells were blocked using blocking buffer (5% v/v normal goat serum in PBS with 0.3% v/v Triton X-100) for one hour at room temperature, before incubation at room temperature for one hour with mouse anti-Flag antibody (Merck, F3165; 1:1,000) in antibody dilution buffer (1% w/v BSA, 0.3% v/v Triton X-100 in PBS). Cells were washed three times with PBS (5 min each), then incubated for one hour at room temperature with goat anti-mouse Alexa488 secondary antibody (Abcam, ab150113; 1:1,000) in antibody dilution buffer. After three final washes in PBS, cells remained in PBS until imaging. Imaging was performed using a Leica TCS SP8 confocal microscope equipped with a water immersion 40× HC PL APO CS2 1.10 NA objective. An OPSL 488 laser (498–622 nm emission) was used to image the anti-mouse AlexaFluor 488 secondary antibody bound to the anti-Flag M2 primary antibody. All transfection conditions were performed in duplicate and three fields of view were captured per well. Representative single-cell images are shown.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
