Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway

Transform the yeast strain genomically expressing Lst1-mCherry with a centromeric plasmid expressing GFP-tagged Gas1 under control of its own promoter (pRS416-GAS1-GFP).

Pick up a transformed colony and streak it on an SD agar plate with appropriate amino acid supplements (SC-URA) and incubate them at 24°C for 2-3 days.

Inoculate transformed yeast cells in 3ml of SC-URA in a 12ml sterile tube and grow them to the early-to-mid logarithmic phase at 24°C with shaking at 250 rpm.

Dilute yeast cells in 200 ml of YPD medium and grow them overnight at 24ºC with shaking at 250 rpm until reaching 1.5 x 10⁷ cells/ml. Note: YPD medium is required for correct cell surface expression of Gas1-GFP.

Collect 120 x 10⁷ cells per sample condition and centrifuge at 3000 x g for 5 min at 4°C.

Discard the supernatant and resuspend by vortexing the cell pellet in 1.5 ml of B88 buffer precooled at 4 °C. Transfer the cell suspension to a 2 ml screw-cap tube.

Centrifuge at 13,000 x g for 1 min at 4 °C, discard supernatant and freeze the pellet at -80ºC.

Quick thaw the cell pellets and immediately place on ice.

Resuspend each cell pellet with 1.5 ml of prechilled B88 buffer.

Add 300 µl of glass beads per tube and lyse mechanically the cells using a bead beater system. For Fastprep device: 3 pulses of 20 s at 5 m/s. Rest on ice for 3 min between pulses.

Spin down glass beads and cell debris at 1000 x g for 10 min at 4°C.

Collect 1 ml of supernatant into a 1.5 ml ultraclear tube.

During the 10 min centrifugation at 4°C at 1000 x g (step 11), prepare a fresh 10x DSP stock solution (10mM DSP in DMSO anhydrous).

Add 110 µl of 10 x DSP stock solution to the cleared cell extract (step 12) and incubate for 20 min at 20ºC in a thermoblock.

Quench the cross-linking reaction by adding 150 µl of 10x glycine stock solution and incubate for 5 min at 20ºC.

Spin down cellular membranes at 13,000 x g for 15 min at 4°C to enrich the pellet with ER membranes.

Discard the supernatant and resuspend each membrane pellet in 1 ml of PBS at 4ºC.

Add 250 µl of PBS-D 5% digitonin to the tubes with the crosslinked samples from step 17 to obtain a solution with a final concentration of 1% digitonin. Mix with end-over-end rotation for 1 h at 4°C.

Centrifuge the cell suspension at 17,000 x g for 20 min at 4°C to remove insoluble membranes.

Transfer the supernatant to a 1.5 ml ultraclear tube and add 100 µl of a 15% solution of naked agarose beads (Bab beads) and rotate for 1 h at 4°C to remove unspecific protein binding.

Centrifuge at 5,000 x g for 30 s and transfer the supernatant to a 1.5 ml ultraclear tube.

Save a 20 μl aliquot of each supernatant for the analysis of protein expression. These will be the total lysate input samples.

Add 100µl of GFP-Trap agarose beads (30% slurry) to the remainder of the supernatant (step 21) and rotate overnight at 4°C.

The next day centrifuge at 5,000 x g for 30 s and remove supernatant.

Resuspend the beads pellet with 1 ml of PBS-D 1% and transfer to a new 1.5 ultraclear tube.

Centrifuge at 5,000 x g for 30 s and remove the supernatant.

Add 1 ml PBS-D 0.2%. Repeat steps 25 and 26 twice more.

Centrifuge at 5,000 x g for 30 s and remove the supernatant. Centrifuge again to remove the remaining liquid and dry the beads using a white gel-loading micropipette tip.

Elute proteins from the beads and dissociate the crosslinked protein complexes by adding 40 μl of 1x SDS-PAGE sample buffer containing fresh 5% 2-mercaptoethanol at 95°C for 10 min.

Vortex and spin down the beads to the maximum speed at room temperature for 1 min and collect the supernatant (IP sample).

Add 20 μl of 2x SDS-PAGE sample buffer containing fresh 5% 2-mercaptoethanol to the tubes containing 20 μl of the total lysate input (T sample) from step 21, vortex and incubate at 95°C for 10 min.

Load the IP samples and their respective T samples onto a 10 % acrylamide gel, separate the proteins by SDS-PAGE gel electrophoresis and transfer them to a nitrocellulose membrane.

Check protein transfer by incubating the nitrocellulose membrane with Ponceau Staining Solution for 1min and washing it with distilled water.

Block the unspecific antibody binding by incubating the membrane in TBS-T + 5% milk for 30 min with shaking.

Blot the crosslinked proteins with anti-RFP (1:500), anti-Sec24 (1:1000), anti-Emp24 (1:1000 ), anti-GFP (1:500), and anti-Pgk1 (1:5000) antibodies sequentially with membrane stripping between blots to detect Lst1-mCherry, Sec24, Pgk1, and Gas1-GFP respectively.

Wash the membrane 3x 3 min in TBS-T.

Incubate 15 min at room temperature in TBS-T + 100 mM mercaptoethanol + 1 ml SDS 20% (heat only the TBS-T and add the mercaptoethanol and the SDS).

Wash 3x 5 min in TBS-T.

Incubate 30 min at room temperature in stripping buffer (20 mM glycine-HCl, pH 2% and 1% SDS).

Wash the membrane 6x 5 min in TBS-T. The stripped membrane is now ready for reproving with another antibody. The stripped membrane can be also kept overnight in TBS-T + 5% milk + azide at 4ºC. In this case, wash the membrane 3x 3 min in TBS-T before reproving with the antibody.

Carry out appropriate binding specificity controls:

A) Non-expressing Gas1-GFP yeast cells.

B) Without crosslinker.

C) Detection of a non-specific protein such as the cytosolic protein Pgk1.