An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

Significance Protein sorting in the secretory pathway is a fundamentally important cellular process, but the clients of a specific cargo sorting machinery remains largely underinvestigated. Here, utilizing a vesicle formation assay to profile proteins associated with vesicles, we identified cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact with GTP-bound Sar1A. We found that two of them, FAM84B and PRRC1, regulate anterograde trafficking. Moreover, we revealed specific clients of two export adaptors, SURF4 and ERGIC53. These analyses demonstrate that our approach is powerful to identify factors that regulate vesicular trafficking and to uncover clients of specific cargo receptors, providing a robust method to reveal insights into the secretory pathway.

Immunofluorescence was performed as described (1). Images were acquired with a Zeiss Axioobserver Z1 microscope system. Quantifications of the total fluorescence of Sec31A and Gogin97 were performed as described using Image J (2). For each experiment, a fixed threshold was manually selected that covers most of the signal on the original gray-scale images and applied to all images. Individual cells were then selected with the free-hand tool and the total above-threshold fluorescence was determined using the measure function. Permeabilized cell assays were performed as described (3).
Immunoprecipation of FAM84B-HA was performed using HEK 293T cells or FAM84B-HA overexpressed HEK293T cells. The cells were resuspended in PBS buffer and then treated with 2mM 4 DSP crosslinker (Thermo fisher Scientific, number:22586) for 30 min at RT. After treatment, unreacted DSP was quenched with 25 mM Tris (pH 7.4) for 15 min. Then the cells were resuspended in lysis buffer containing 50 mM Tris-HCl, 150mM NaCl, 2mM CaCl2, 1%TX-100, protease inhibitors and 1mM DTT (pH 7.5) on ice for 30min. Anti-HA agarose beads (Thermo fisher Scientific, number:26181) were pre-blocked with blocking buffer (50mM Tris-HCl, 500mM NaCl, 2mM CaCl2, 5% fat-free BSA, pH7.5) at 4 °C with rotation for 1 hr and then incubated with cell lysates for 4°C with rotation overnight. The beads were then washed with blocking buffer for 3 times and followed with lysis buffer for 3 times. Then the HA agarose beads were boiled in reduced sample buffer and analyzed by immunoblot.
Immunoprecipation of PRRC1-FLAG was performed using HEK293T cells or PRRC1-FLAG overexpressed HEK293T cells. The cells were lysed with KOAc buffer containing 0.5%Triton X-100, protease inhibitors and 1mM DTT for 30 min on ice. After 10min 14000 g centrifugation, the cell lysate supernatant was incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich, number A2220) at 4 °C with rotation overnight. The beads were then washed with KOAc buffer and eluted with KOAc buffer containing 0.6 mg/mL 3x FLAG peptides. The eluted fraction and on beads fraction were analyzed by immunoblot.

In vitro vesicle formation assay
In vitro vesicle formation assay was performed as described (1). HEK293T, HEK 293Trex, HEK (Beckman) or S55S (Hitachi) rotor at 4°C for 1.5 hr. After centrifugation, the top fraction (the vesicle fraction) was collected and analyzed by negative stain electron microscope, Coomassie staining, western blot or mass spectrometry.

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Vesicle immuno-gold labeling and negative staining for transmission electron microscopy analysis Negative staining transmission electron microscopy (TEM) analysis was performed essentially as described previously (4). Briefly, 10 µl of the vesicle fraction was applied onto the pre-glow discharged carbon-coated 400-mesh copper grids (EMR, 22-1MC040-50) for 10 min before further labelling or negative staining. The glow discharge was performed using the PELCO easiGlow TM Glow Discharge Cleaning System (Ted Pella, Inc., Redding, CA). For negative staining, extra liquid was removed by filter paper after 10 min vesicle sedimentation on the grid, followed by negatively stained by 10 µl 2% uranyl acetate (UA) for 2 min. Extra UA liquid was removed and the grids were further air dried before TEM observation. For immuno-gold labelling, vesicles on grid were first blocked by 0.1-1% BSA in PBS, followed by primary antibody incubation at optimized concentrations for 2-3 hr. The grids were briefly washed by 0.1% BSA before applying goat antirabbit 6 nm gold-coupled secondary antibodies (EMS, 25104) at 1:20 dilution for 1 hr. Immunogoldlabelled vesicles were then negatively stained as previously described before TEM observation. The quantification and comparison of vesicle size or number were performed in a same area size for images from different groups for each experiment as indicated.

Protein purifications, nucleotide loading and GST pull down
To purify His-tagged proteins from E. coli, cells expressing His-tagged constructs were grown to the optical density at 600 nm (OD600) at 0.6-0.8 in lysogeny broth (LB) and induced with 0.5mM Purification of GST-tagged protein was performed as described (5). The nucleotide loading and GST pull down experiment was performed as described (5,6). Briefly, 10 µL glutathione beads buffer containing 500 µM GMPPNP or GDP at 37 °C for 45 min. After incubation, the beads were incubated with 2 mg/mL RLC containing 100 µM GDP or GMPPNP at 4 °C for 2hrs. The beads were then washed six times with 500 µL NS buffer and one time with 500 µL NS buffer without magnesium and then the bound proteins were eluted with elution buffer (20mM Hepes, 1.5M KCl, 0.5% Triton-100, 20mM EDTA) and analyzed by immunoblot. All of the incubations were performed in the presence of proteinase inhibitors.
The same volume of 8M urea dissolved in 50 mM TEAB buffer was added into the vesicle sample.
The sample was then reduced with 10mM TCEP at 37°C for 1 hr and alkylated with 20mM iodoacetamide (IAA) at room temperature in the dark for 30 min, followed by the dilution with 50 mM TEAB for 4 times and the digestion with sequencing grade modified trypsin (Promega, number V511A) at 37 °C for 20 hr. To stop the trypsin digestion and to remove the surfactant, the pH of vesicle samples was adjusted to 2.5-3.0 by adding 10% trifluoroacetic acid (TFA). The degraded surfactant was removed by centrifugation. Samples were dried by speed vacuum. Next, samples were desalted with pierce C18 spin column (Thermo fisher Scientific, number 89870). Subsequently, the samples were dried again to be analyzed by the mass spectrometry.

Liquid chromatography-MS analysis
Mass spectrometry and data analysis was performed as described (7). Briefly, LC separation was performed using an Acclaim PepMap RSLC C18 capillary column (75 μm × 25 cm; 2 μm particles, 100 Å) (Thermo fisher Scientific, San Jose, CA). Gradient elution was performed using an Ultimate 3000 nanoLC system (Thermo fisher Scientific, San Jose, CA). The flow rate was set at 300 nl/min.
Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The analytical gradient lasted for 90 min including the following steps: 1) 10 min of equilibration with 3% B; 2) the composition of solvent B was increased from 3% to 7% in 2 min, from 7% to 20% in 50 min, and from 20% to 30% in 2 min; 3) a washing and equilibration step when solvent B was increased to 80% in 1 min and was held for 8 min; 4) the composition of solvent B was returned to 3% in 0.1 min and was held for 17 min.
Analysis was performed using an Orbitrap Fusion Lumos mass spectrometer (Thermo fisher Scientific, San Jose, CA) operating in positive ion mode. The ESI spray voltage was set at 2300 KV and the ion transfer tube temperature was set at 300°C. MS and MS/MS scans were performed using 7 high resolution Orbitrap, with resolution at 60K and 15K, respectively. Data-dependent acquisition (DDA) mode was performed with a cycle time of 3 s. The mass range of the full MS scan defines m/z 400 to 1600, and in MS/MS starts from m/z 110. The collision energy was set at 30%. Three biological repeats of each sample were performed.

MS data analysis
Proteome Discoverer 2.2.0.388 was used for protein identification and quantification with the following settings: (1) fixed modification: cysteine carbamidomethylation (+57.021 Da); (2) dynamic modification: methionine oxidation (+15.995 Da) and acetylation (+42.011) at the N terminus of the protein; (3) trypsin was used for digestion with one missed cleavage allowed; (4) peptide ion tolerance: 10 ppm; (5) fragment ion tolerance: 20 ppm; (6) the protein sequence database of Homo sapiens was downloaded from Uniprot (updated 11-2018) for database searching and identification with a false discovery rate (FDR) <0.01; (7) the minora algorithm based label-free quantification (LFQ) was performed using the intensity of precursor ions; (8) unique and razor peptides of proteins were selected to calculate the abundance ratio of proteins; (9)The abundance ratio of each identified proteins was determined using pairwise-ratio based calculation where the median value of the abundance ratio of all matched peptides from three biological repeats was used as the abundance ratio of the identified protein and the maximum allowed fold change was set as 100; (10) The protein abundance was determined by the average intensities of top three unique peptides (if <3 peptides can be quantified, the average intensity of these peptides was used).
Proteins with two or more unique peptides and successfully quantified in all of the three biological repeats of at least one experimental group were selected for quantitative analysis. The protein abundance was normalized to the median value of each sample to correct experimental bias based on the total protein amount before further statistical comparison. (8) HEK293 Flp-In Trex WT, SURF4 KO and LMAN1 KO cells were seeded at 5000 cells/well in a 96-well plate (Corning, 3610). The following day cells were transfected in replicates of 6 with a plasmid containing 5 tandem ATF6 promoter elements upstream of Renilla Firefly luciferase ORF (8). The next day control wells were treated with the following UPR inducers: tunicamycin (2.5 ug/mL), thapsigargin (0.2 uM) and increasing concentrations of dithiothreitol (DTT) (0.2-1 mM).

ATF6-Luciferase Unfolded Protein Response (UPR) assay
After 16h of antibiotic treatment media in all wells was aspirated, exchanged for complete DMEM containing 100 uM Luciferin (Carbosynth, L-8220), incubated at 37C for 30 min and assayed using a luminescence plate reader.       siRNA or siRNA against SURF4 (SURF4 KD). On day 3 after transfection, the vesicle formation was performed using the indicated cells and reagents. Vesicle fractions were then analyzed by immunoblot. C-D. Quantifications of the percentage of NUCB1 or NUCB2 that was packaged into transport vesicles (n = 3, mean ± S.D.). *, p < 0.05; **, p < 0.01. E. Cell lysates from HEK293T cells transfected with N.C. siRNA or siRNA against SURF4 were analyzed by immunoblot. F. HEK293T cells expressing SURF4-HA were treated in 2mM DSP, and the cell lysates were incubated with beads conjugated with HA antibodies. After incubation, the bound proteins were eluted analyzed by western blot using the indicated antibodies.