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†, Alejandro Cortes-Gomez, 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 stabilize 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 transformation 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: Yeast transformation and culture
2. Pick up a transformed colony and streak it on an SC-URA agar plate and incubate them at 24°C for 2-3 days.
SECTION: Yeast transformation and culture
3. Inoculate transformed yeast cells in 3 ml of SC-URA in a 12 ml sterile tube and grow them to the early-to-mid logarithmic phase at 24°C with shaking at 250 rpm.
SECTION: Yeast transformation 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 transformation and culture
5. Collect 240 x 107 cells per sample condition and centrifuge at 3000 x g for 5 min at 4°C.
SECTION: Yeast transformation 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 transformation and culture
7. Centrifuge at 13,000 x g for 1 min at 4°C, discard the supernatant and freeze the pellet at -80ºC.
SECTION: Cell lysis
8. Quick thaw the cell pellets and immediately place them 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: Cell lysis
12. Collect 1 ml of supernatant into a 1.5 ml ultraclear tube.
SECTION: Cross-linking
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: Cross-linking
14. Add 110 µl of 10x DSP stock solution to the cleared cell extract (step 12) and incubate for 20 min at 20ºC in a thermoblock.
SECTION: Cross-linking
15. Quench the cross-linking reaction by adding 150 µl of 10x glycine stock solution and incubate for 5 min at 20ºC.
SECTION: Cross-linking
16. Spin down cellular membranes at 13,000 x g for 15 min at 4°C to enrich the pellet with ER membranes.
SECTION: Cross-linking
17. Discard the supernatant and resuspend each membrane pellet in 1 ml of PBS at 4ºC.
SECTION: co-immunoprecipitation of cross-linked proteins
18. Centrifuge the cell suspension at 17,000 x g for 20 min at 4°C to remove insoluble membranes.
SECTION: co-immunoprecipitation of cross-linked proteins
19. 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 cross-linked proteins
20. Centrifuge at 5,000 x g for 30 s and transfer the supernatant to a 1.5 ml ultraclear tube.
SECTION: co-immunoprecipitation of cross-linked proteins
21. 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 cross-linked proteins
22. 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 cross-linked proteins
23. The next day centrifuge at 5,000 x g for 30 s and remove supernatant.
SECTION: co-immunoprecipitation of cross-linked proteins
24. Resuspend the beads pellet with 1 ml of PBS-D 1% and transfer to a new 1.5 ultraclear tube.
SECTION: co-immunoprecipitation of cross-linked proteins
25. Centrifuge at 5,000 x g for 30 s and remove the supernatant.
SECTION: co-immunoprecipitation of cross-linked proteins
26. Add 1 ml PBS-D 0.2%. Repeat steps 25 and 26 twice more.
SECTION: co-immunoprecipitation of cross-linked proteins
27. 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 cross-linked proteins
29. 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 cross-linked proteins
30. 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
31. Load IP samples (20 μl) with their respective T samples (10 μl) onto a 10% acrylamide gel. Separate the proteins by SDS-PAGE gel electrophoresis and transfer them to a nitrocellulose membrane.
SECTION: Western blotting
32. To check protein transfer, stain the nitrocellulose membrane in Ponceau Staining Solution for 3 min and wash with distilled water.
SECTION: Western blotting
33. Block the membranes in TBS-T + 5% milk for 1 h at room temperature with shaking.
SECTION: Western blotting
34. 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 (~132 kD), Sec24 (~104 kD), Pgk1 (~45 kD), and Gas1-GFP (~135 kD) respectively. Alternatively,  since all these antibodies are highly specific, the total number of strippings can be reduced by cutting the membrane horizontally after protein transfer and probing each separate membrane portion with the different antibodies. In this particular case, cut the membrane horizontally at 75 kD and 25 kD prestained protein markers in three portions. Probe sequentially the top portion for Lst1-mCherry, Sec24, and Gas1-GFP with membrane stripping between the blots. Probe medium and bottom portions for Pgk1 and Emp24 respectively. In addition to reducing the total number of strippings, this also saves limited primary antibodies.
SECTION: Western blotting
35. Incubate the membranes in primary antibody diluted in TBS-T + 5% milk 1 h at room temperature or overnight at 4°C, with shaking.
SECTION: Western blotting
36. Wash the membranes in TBS-T + 5% milk, 5x 5 min.
SECTION: Western blotting
37. Incubate the membranes in HRP-conjugated secondary antibody diluted in TBS-T + 5% milk 1 h at room temperature with shaking.
SECTION: Western blotting
38. Wash the membranes in TBS-T 5x 5 min.
SECTION: Western blotting
39. Visualize crosslinked proteins by using enhanced chemiluminescence (ECL) detection through the Bio-Rad ChemiDoc instrument.
SECTION: Membrane stripping for antibody reprobing
40. Wash the membranes 3x 3 min in TBS-T.
SECTION: Membrane stripping for antibody reprobing
41. Incubate the membranes for 15 min at room temperature in TBS-T + 100 mM 2-mercaptoethanol + 2% SDS.
SECTION: Membrane stripping for antibody reprobing
42. Wash the membranes 3x 5 min in TBS-T.
SECTION: Membrane stripping for antibody reprobing
43. Incubate the membranes for 30 min at room temperature in stripping buffer.
SECTION: Membrane stripping for antibody reprobing
44. Wash the membranes 6x 5 min in TBS-T. The stripped membranes are now ready for reprobing with another antibody (step 35). The stripped membrane can be also kept overnight in TBS-T + 5% milk + 0.01% sodium azide at 4ºC. In this case, wash the membrane 3x 3 min in TBS-T before reproving with the antibody.
SECTION: Membrane stripping for antibody reprobing
45. Block the membranes in TBS-T + 5% milk for 1 h at room temperature with shaking. 
SECTION: Membrane stripping for antibody reprobing
46. Reprobe the stripped membranes with another antibody (step 36).
SECTION: Controls for binding specificity
47. Carry out appropriate binding specificity controls:
a) Wild-type yeast cells expressing no Gas1-GFP. 
b) Without crosslinker. 
c) Detection of a non-specific protein such as the cytosolic protein Pgk1.
SECTION: Yeast transformation and culture
1.1. Grow a 5 ml overnight culture of the yeast strain to be transformed at 24ºC with shaking at 250 rpm.
SECTION: Yeast transformation and culture
1.2. The next day, collect 5 x107 cells into a 15 ml tube and centrifuge at 3,000 x g for 5 min at room temperature.
SECTION: Yeast transformation and culture
1.3. Discard the supernatant, resuspend the cell pellet in 1 ml of sterile dH2O, transfer into a 1.5 ml tube, and centrifuge at 3,000 x g for 5 min at room temperature.
SECTION: Yeast transformation and culture
1.4. Discard the supernatant and resuspend the cell pellet in 100 µl with “One-step” buffer.
SECTION: Yeast transformation and culture
1.5. Add 1 µg of plasmid DNA (pRS416-GAS1-GFP) and 50 µg of boiled salmon sperm DNA. Briefly vortex.
SECTION: Yeast transformation and culture
1.6. Incubate 30-40 min at 45°C and plate on appropriate selection media (SC-URA).
SECTION: Yeast transformation and culture
1.7. Note: Carry out a negative control sample without DNA plasmid.
SECTION: co-immunoprecipitation of cross-linked proteins
28. Elute proteins from the beads and dissociate the crosslinked protein complexes by adding 40μl of 1x SDS-PAGE sample buffer containing fresh 5% 2-mercaptoethanol at 95°C for 10 min.