TITLE: Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway
AUTHORS: Javier              Manzano-Lopez †, Sofia Rodriguez-Gallardo †, Susana              Sabido-Bozo, Ana              Maria Perez-Linero, Rafael             Lucena, Antonio              Cordones-Romero, Sergio Lopez, Auxiliadora              Aguilera-Romero, Manuel  Muñiz

[DESCRIPTION]
Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

[STEPS]
SECTION: Yeast growth and culture
1. 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).
SECTION: Cell lysis
8. Quick thaw the cell pellets and immediately place on ice.
SECTION: Cell lysis
9. Resuspend each cell pellet with 1.5 ml of prechilled B88 buffer.
SECTION: Cell lysis
10. 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.
SECTION: Cell lysis
11. Spin down glass beads and cell debris at 1000 x g for 10 min at 4°C.
SECTION: Crosslinking
13. 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).
SECTION: Crosslinking
14. 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.
SECTION: Crosslinking
15. Quench the cross-linking reaction by adding 150 µl of 10x glycine stock solution and incubate for 5 min at 20ºC.
SECTION: Crosslinking
16. Spin down cellular membranes at 13,000 x g for 15 min at 4°C to enrich the pellet with ER membranes.
SECTION: Crosslinking
17. Discard the supernatant and resuspend each membrane pellet in 1 ml of PBS at 4ºC.
SECTION: Co-immunoprecipitation of crosslinked proteins
18. 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.
SECTION: Co-immunoprecipitation of crosslinked proteins
20. 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.
SECTION: Co-immunoprecipitation of crosslinked proteins
21. Centrifuge at 5,000 x g for 30 s and transfer the supernatant to a 1.5 ml ultraclear tube.
SECTION: Co-immunoprecipitation of crosslinked proteins
19. Centrifuge the cell suspension at 17,000 x g for 20 min at 4°C to remove insoluble membranes.
SECTION: Co-immunoprecipitation of crosslinked proteins
22. Save a 20 μl aliquot of each supernatant for the analysis of protein expression. These will be the total lysate input samples.
SECTION: Co-immunoprecipitation of crosslinked proteins
23. Add 100µl of GFP-Trap agarose beads (30% slurry) to the remainder of the supernatant (step 21) and rotate overnight at 4°C.
SECTION: Co-immunoprecipitation of crosslinked proteins
24. The next day centrifuge at 5,000 x g for 30 s and remove supernatant.
SECTION: Co-immunoprecipitation of crosslinked proteins
25. Resuspend the beads pellet with 1 ml of PBS-D 1% and transfer to a new 1.5 ultraclear tube.
SECTION: Co-immunoprecipitation of crosslinked proteins
26. Centrifuge at 5,000 x g for 30 s and remove the supernatant.
SECTION: Co-immunoprecipitation of crosslinked proteins
27. Add 1 ml PBS-D 0.2%. Repeat steps 25 and 26 twice more.
SECTION: Co-immunoprecipitation of crosslinked proteins
28. 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.
SECTION: Co-immunoprecipitation of crosslinked proteins
29. 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.
SECTION: Co-immunoprecipitation of crosslinked proteins
30. Vortex and spin down the beads to the maximum speed at room temperature for 1 min and collect the supernatant (IP sample).
SECTION: Co-immunoprecipitation of crosslinked proteins
31. 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.
SECTION: Western blotting
32. 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.
SECTION: Western blotting
35. 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.
SECTION: Stripping
36. Wash the membrane 3x 3 min in TBS-T.
SECTION: Stripping
37. 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).
SECTION: Stripping
38. Wash 3x 5 min in TBS-T.
SECTION: Stripping
39. Incubate 30 min at room temperature in stripping buffer (20 mM glycine-HCl, pH 2% and 1% SDS).
SECTION: Stripping
40. 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.
SECTION: Controls
41. 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.
SECTION: Yeast growth and culture
2. 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.
SECTION: Yeast growth and culture
3. 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.
SECTION: Yeast growth and culture
4. 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.
SECTION: Yeast growth and culture
5. Collect 120 x 107 cells per sample condition and centrifuge at 3000 x g for 5 min at 4°C.
SECTION: Yeast growth and culture
6. 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.
SECTION: Yeast growth and culture
7. Centrifuge at 13,000 x g for 1 min at 4 °C, discard supernatant and freeze the pellet at -80ºC.
SECTION: Cell lysis
12. Collect 1 ml of supernatant into a 1.5 ml ultraclear tube.
SECTION: Western blotting
33. Check protein transfer by incubating the nitrocellulose membrane with Ponceau Staining Solution for 1min and washing it with distilled water.
SECTION: Western blotting
34. Block the unspecific antibody binding by incubating the membrane in TBS-T + 5% milk for 30 min with shaking.