Materials and Reagents

1. 1.5 mL tube

2. 250 mL flask

3. NHS beads (TAMAGAWA SEIKI, catalog number: TAS8848N1141)

4. Anti-DYKDDDDK antibodies (WAKO, catalog number: 1E6)

5. Methanol (Fisher Scientific, catalog number: A454-4)

6. 250 mM MES-NaOH (pH 6.0) (Sigma, catalog number: 69892)

7. 1 M Ethanolamine (Fisher Scientific, catalog number: AC149582500)

8. 0.1 M Glycine-HCl (pH 2.5) (VWR, catalog number: 0617-5KG)

9. 1.0 M Tris-HCl (pH 8.0) (VWR, catalog number: 95029-578)

10. Protease inhibitor (Complete Mini EDTA-free) (Roche, catalog number: 04693159001)

11. 0.5 mm Zirconia/Silica beads (Biospec Products, catalog number: 11079105Z)

12. HEPES-NaOH (pH 7.4) (VWR, catalog number: EM-5330)

13. KCl (Fisher Scientific, catalog number: P330-500)

14. EDTA (Sigma, catalog number: E9884)

15. Glycerol (Fisher Scientific, catalog number: G33-1)

16. Triton X-100 (Sigma, catalog number: 93443)

17. BSA (Sigma, catalog number: A9418)

18. Sodium azide (Sigma, catalog number: S2002)

19. Sorbitol (VWR, catalog number: 97062-204)

20. SDS (Sigma, catalog number: L3771)

21. DTT (Santa Cruz Biotechnology)

22. Bromophenol blue (Sigma, catalog number: 115-39-9)

23. Yeast extract (BD, catalog number: 210929)

24. Bacto peptone (BD, catalog number: 211677)

25. Glucose (VWR, catalog number: BDH9230)

26. Agar (VWR, catalog number: 244510)

27. Protein conjugation buffer (see Recipes)

28. Protein conjugation buffer (+0.5% Triton X-100) (see Recipes)

29. Protein storage buffer (see Recipes)

30. YEPD plate (see Recipes)

31. YEPD liquid culture (see Recipes)

32. H₂₅S₇₅E₅ buffer (see Recipes)

33. H₂₅S₇₅E₅ buffer (+PIC) (see Recipes)

34. SDS-PAGE sample buffer (see Recipes)

Strains

1. SEY 6210 sec18^ts yeast cells expressing VPS10-3xFLAG (Suzuki et al., 2021)

2. SEY 6210 sec18^ts yeast cells expressing VPS10-3xFLAG (Suzuki et al., 2021)

Equipment

1. Microcentrifuge (Fisher Scientific, model: AccuSpin Micro 17/17R)

2. Centrifuge (Beckman Coulter, model: Allegra X-15R)

3. Ultrasonic Cleaner (Fisher Scientific, model: FS30)

4. Micro Tube Mixer (Tomy, model: MT-360)

5. Labquake^TM Tube Shaker/Rotators (Thermo Scientific)

6. Magnetic stand (TAMAGAWA SEIKI, model: TAB4899N12)

7. Labquake Tube Shaker/Rotators (Thermo Scientific, model: 415110Q)

8. SmartSpec Plus Spectrophotometer (BIO-RAD)

9. Mini Vortexer (VWR)

10. Dry Block Incubator (Fisher Scientific)

11. 1545 VWR Digital Incubator (VWR)

12. New Brunswick^TM Innova^® 44/44R Stackable Incubator Shakers (Eppendorf)

Procedure

1. Immobilization of anti-DYKDDDDK antibody on NHS beads

   1. Transfer 5.0 mg of NHS beads into a 1.5-mL tube.

   2. Centrifuge the beads at 15,000 × g and 4°C for 5 min, and remove the supernatant.

   3. Resuspend the beads in 250 μL of methanol, and disperse them by sonication (less than 1 min).

   4. Centrifuge the beads at 15,000 × g and 4°C for 5 min, and remove the supernatant.

   5. Resuspend the beads in 160 μL of 25 mM MES-NaOH (pH 6.0), and disperse them by sonication.

   6. Add 160 μL of anti-DYKDDDDK antibodies in 25 mM MES-NaOH (pH 6.0).

      Note: Resuspend 0.5 mg of anti-DYKDDDDK antibodies in 160 μL of 25 mM MES-NaOH (pH 6.0).

   7. Incubate the beads with gentle vortexing, using a microtube mixer at 4°C for 30 min.

      Note: Set the dial of the microtube mixer to 6.5.

   8. Collect the beads using a magnetic stand, and remove the supernatant.

      Note: Spin the tube down before collecting the beads, to collect liquid at the lip of tube.

   9. Resuspend the beads in 625 μL of 1 M ethanolamine, and disperse the beads by pipetting.

   10. Incubate the beads using the microtube mixer at 4°C overnight.

   11. Collect the beads using a magnetic stand, and remove the supernatant.

   12. Resuspend the beads in 1 mL of protein conjugation buffer, and disperse the beads by pipetting.

   13. Collect the beads using a magnetic stand, and remove the supernatant.

   14. Repeat steps A12 and A13 three times.

   15. Resuspend the beads in 1 mL of 0.1 M Glycine-HCl (pH 2.5).

   16. Collect the beads using a magnetic stand, and remove the supernatant.

   17. Resuspend the beads 1 mL of 1.0 M Tris-HCl (pH 8.0).

   18. Collect the beads using a magnetic stand, and remove the supernatant.

   19. Resuspend the beads in 1 mL of protein conjugation buffer (+0.5% Triton X-100).

   20. Incubate the beads with gentle rotation using a tube shaker/rotator at 4°C for 15 min.

   21. Collect the beads using a magnetic stand, and remove the supernatant.

   22. Resuspend the beads in 1 mL of protein conjugation buffer.

   23. Collect the beads using a magnetic stand, and remove the supernatant.

   24. Repeat steps A22 and A23 three times.

   25. Resuspend the beads in 1 mL of protein storage buffer, and store at 4°C.

       Note: A volume of 1 mL of beads can be used for 100 experiments. You can store the beads at 4°C for at least one year.

       
       

2. Immunoisolation of recycling vesicles

   1. Streak Vps55-3xFLAG or Vps10-3xFLAG expressing sec18^ts yeast strains to the YEPD plate from glycerol stocks (yeasts in 15% glycerol and YEPD, store at -80°C).

      Note: To purify retromer-recycling vesicles, Vps10-3xFLAG sec18^ts cells are used. For Mvp1-recycling vesicles, Vps55-3xFLAG sec18ts cells are used.

   2. Incubate the plate in a VWR digital incubator at 26°C for three days.

   3. Inoculate the yeast cells in 5 mL of YEPD liquid medium and cultivate them in incubator shakers at 250 rpm and 26°C overnight.

   4. Inoculate 0.5–2.5 μL of the preculture to 5 mL of YEPD liquid culture. Inoculate this to 100 mL of YEPD liquid media in a 250-mL flask and grow the cells in incubator shakers at 250 rpm and 26°C overnight.

   5. When the cells reach mid-log phase (OD₆₀₀ = 0.4–0.8), incubate them at in incubator shakers at 250 rpm and 37°C for 60 min.

      Note: Measure OD₆₀₀ of the undiluted yeast culture by pipetting 100 μL of the yeast culture into 900 μL of water.

   6. Cool the cells on ice for 5 min.

   7. Harvest 80 OD₆₀₀ unit yeast cells into a 50-mL tube, and centrifuge it using the centrifuge (Beckman Coulter, Allegra X-15R) at 4,000 × g and 4°C for 2 min.

   8. Remove the supernatant by pipetting, and resuspend the pellet in 5 mL of cooled H₂₅S₇₅E₅ buffer.

   9. Centrifuge the cells at 4,000 × g and 4°C for 2 min, and remove the supernatant by pipetting.

   10. Resuspend the pellet in 1 mL of cooled H₂₅S₇₅E₅ buffer into a 1.5-mL micro-tube.

   11. Centrifuge the cells at 10,000 × g for 1 min, and remove the supernatant.

   12. Resuspend the cells in 1 mL of cooled H₂₅S₇₅E₅ buffer (+PIC), and add 500 μL of 0.5 mm Zirconia/Silica beads.

       Note: Buffer should not contain detergent, but does contain 750 mM sorbitol to maintain osmolarity.

   13. Vortex the cells to lyse them at 4°C for 1 min.

   14. Centrifuge the lysate in a 1.5-mL micro-tube at 500 × g and 4°C for 5 min, and remove the supernatant.

   15. Centrifuge the supernatant at 15,000 × g and 4°C for 10 min, and remove the supernatant.

   16. Incubate the cleared supernatant with 10 μL of pre-equilibrated anti-DYKDDDDK NHS beads (prepared in “A”) with gentle rotation using tube shaker/rotators at 4°C for 60 min.

       Note: Before use, suspend anti-DYKDDDDK NHS beads in cooled H₂₅S₇₅E₅ buffer.

   17. Collect the beads using a magnetic stand, and remove the supernatant by pipetting.

   18. Resuspend the beads in 1 mL of cooled H₂₅S₇₅E₅ buffer.

   19. Repeat steps B18 and B19 three times.

   20. Transfer the beads to a new 1.5-mL tube.

   21. Collect the beads using a magnetic stand, and remove the supernatant by pipetting.

   22. Add 100 μL of SDS-PAGE sample buffer.

   23. Incubate the beads at 98°C for 5 min.

   24. Collect the beads using a magnetic stand, and remove the supernatant.

   25. Analyze the sample by SDS-PAGE and western blotting.

       
       

   
   Figure 2. Immunoisolation of endosomal recycling vesicles. A. Schematic for immunoisolation of endosomal recycling vesicles. To accumulate cargo-containing recycling vesicles, a temperature-sensitive mutant of Sec18, which is essential for the fusion of recycling vesicles with the Golgi apparatus, was used. After 60 min of incubation at the non-permissive temperature (37°C), cells expressing FLAG-fused cargo proteins (Vps10 or Vps55) were lysed. Then, cargo-containing recycling vesicles were immunoisolated by anti-FLAG magnetic beads. B. Western blotting analysis of the isolated Vps55-FLAG-containing vesicles. C. Electron microscopy (EM) analysis of the isolated Vps55-FLAG-containing vesicles. Data reproduced with modification for space limits from Suzuki et al. (2021).

Recipes

1. Protein conjugation buffer

   10 mM HEPES-NaOH (pH 7.4)

   50 mM KCl

   1 mM EDTA

   10% Glycerol

   Note: Protein conjugation buffer can be stored at room temperature (RT).

2. Protein conjugation buffer (+0.5% Triton X-100)

   10 mM HEPES-NaOH (pH 7.4)

   50 mM KCl

   1 mM EDTA

   10% Glycerol

   0.5% Triton X-100

   Note: Protein conjugation buffer (+0.5% Triton X-100) can be stored at RT.

3. Protein storage buffer

   10 mM HEPES-NaOH (pH 7.4)

   50 mM KCl

   1 mM EDTA

   10% Glycerol

   1 mg/mL BSA

   3 mM Sodium azide

   Note: Protein storage buffer can be stored at RT.

4. H₂₅S₇₅E₅ buffer

   25 mM HEPES-NaOH (pH 7.4)

   750 mM Sorbitol

   5 mM EDTA

   Note: H₂₅S₇₅E₅ buffer can be stored at RT.

5. H₂₅S₇₅E₅ buffer (+PIC)

   25 mM HEPES-NaOH (pH 7.4)

   750 mM Sorbitol

   5 mM EDTA

   1× Protease inhibitor cocktail

   Note: H₂₅S₇₅E₅ buffer (+PIC) needs to be freshly prepared.

6. SDS-PAGE sample buffer

   75 mM Tris-HCl (pH 7.5)

   2% (w/v) SDS

   10% Glycerol

   100 mM DTT

   Bromophenol blue

   Note: SDS-PAGE sample buffer needs to be freshly prepared.

7. YEPD plate

   1% (w/v) Yeast extract

   2% (w/v) Bacto peptone

   2% Glucose

   2% Agar

8. YEPD liquid culture

   1% (w/v) Yeast extract

   2% (w/v) Bacto peptone

   2% Glucose

Acknowledgments

SW Suzuki is supported by JSPS Postdoctoral Fellowships for Research Abroad and the Osamu Hayaishi Memorial Scholarship for Study Abroad. This work was supported by a Cornell University Research Grant (CU563704) to SD Emr. This protocol is based on Suzuki et al. (2021).

Competing interests

The authors declare that no competing interests exist.

References

1. Hettema, E. H., Lewis, M. J., Black, M. W. and Pelham, H. R. (2003). Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes. EMBO J 22(3): 548-557.
2. Henne, W. M., Buchkovich, N. J. and Emr, S. D. (2011). The ESCRT pathway. Dev Cell 21(1): 77-91.
3. Seaman, M. N., McCaffery, J. M. and Emr, S. D. (1998). A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol 142(3): 665-681.
4. Suzuki, S. W., Oishi, A., Nikulin, N., Jorgensen, J. R., Baile, M. G. and Emr, S. D. (2021). A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling. Elife 10: e69883.