Identification of the Subcompartment-Specific Mitochondrial Proteome by APEX2 Proximity Labeling in Saccharomyces cerevisiae

Materials and reagents

Biological materials

1. Saccharomyces cerevisiae W303 strain (MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [phi+]) (Ralser et al. [17])

2. Saccharomyces cerevisiae W303 strain + Su9-APEX2-FLAG (YPRC∆15-pRNR1- Mia40(1-70)-APEX2-FLAG-tTDH1-HygromycinR) (see Appendix in [15])

3. Saccharomyces cerevisiae W303 strain + IMS-APEX2-FLAG (YPRCD15-pRNR1-Su9-APEX2-FLAG-tTDH1-HygromycinR) (see Appendix in [15])

Note: Yeast strains and plasmids used for integration are available upon request.

Reagents

1. Adenine hemisulfate salt (Sigma-Aldrich, catalog number: A3159)

2. L-Arginine (Sigma-Aldrich, catalog number: A8094)

3. L-Histidine monohydrochloride monohydrate (Sigma-Aldrich, catalog number: H5659)

4. L-Isoleucine (Sigma-Aldrich, catalog number: I7403)

5. L-Leucin (Sigma-Aldrich, catalog number: L8000)

6. L-Lysin HCl (Sigma-Aldrich, catalog number: L8662)

7. L-Methionine (Sigma-Aldrich, catalog number: M5308)

8. L-Phenylalanine (Sigma-Aldrich, catalog number: P2126)

9. L-Threonine (Sigma-Aldrich, catalog number: T8441)

10. L-Tryptophan (Sigma-Aldrich, catalog number: T8941)

11. L-Tyrosine (Sigma-Aldrich, catalog number: T8566)

12. Uracil (Sigma-Aldrich, catalog number: U1128)

13. L-Valine (Sigma-Aldrich, catalog number: V0513)

14. L-Proline (Sigma-Aldrich, catalog number: P5607)

15. Yeast-nitrogen-base (Formedium, catalog number: CYN0510)

16. Yeast-nitrogen-base w/o biotin (Formedium, catalog number: CYN3202)

17. Ammonium sulfate (Sigma-Aldrich, catalog number: 1.01217)

18. Galactose (Sigma-Aldrich, catalog number: G0625)

19. Dithiothreitol (DTT) (AppliChem, catalog number: A1101)

20. Sorbitol (Carl Roth, catalog number: 6213.2)

21. KH₂PO₄ (Bernd Kraft, catalog number: 15258.3600)

22. K₂HPO₄ (Carl Roth, catalog number: 7758-11-4)

23. Tris(hydroxymethyl)aminomethane (Serva, catalog number: 77-81-1)

24. HCl 37% (VWR Chemicals, catalog number: 20252.335)

25. Zymolyase (Carl Roth, catalog number: 9324.3)

26. EDTA disodium salt 2-hydrate (AppliChem, catalog number: 131669)

27. Albumin bovine fraction V, fatty acid free (BSA) (Serva, catalog number: 11932.03)

28. Phenylmethylsulphonyl fluoride (PMSF) (Carl Roth, catalog number: 6367.2)

29. Protein assay dye (Bio-Rad Laboratories, catalog number: 5000006)

30. Liquid N₂

31. Biotin-phenol (Sigma-Aldrich, catalog number: SML2135)

32. Dimethyl sulfoxide (DMSO) (VWR Chemicals, catalog number: 23500.397)

33. H₂O₂ 30% (Sigma-Aldrich, catalog number: 216763)

34. Ascorbic acid (AppliChem, catalog number: A3604)

35. Sodium azide (Merck, catalog number: 8.22335)

36. Trolox (Thermo Scientific, catalog number: 218940050)

37. NaH₂PO₄·H₂O (Merck, catalog number: 1.06346)

38. Na₂HPO₄ (Sigma-Aldrich, catalog number: 71640)

39. NaCl (Fisher Scientific, catalog number: 10616082)

40. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896)

41. Deoxycholic acid sodium salt (Carl Roth, catalog number: 3484.2)

42. Sodium dodecyl sulfate (SDS) (Serva, catalog number: 20765.03)

43. Protease inhibitor (cOmplete tablets) (Roche, catalog number: 04693159001

44. Pierce streptavidin magnetic beads (Thermo Scientific, catalog number: 88817)

45. Urea (Serva, catalog number: 24524.0)

46. Trypsin (Promega, catalog number: V5111)

47. Trypsin resuspension buffer (Promega, catalog number: V5111)

48. Chloroacetamide (CAA) (Sigma-Aldrich, catalog number: C0267)

49. Formic acid (Honeywell, catalog number: 94318)

50. Acetonitrile (VWR Chemicals, catalog number: 83640.290)

51. MS-grade H₂O (VWR Chemicals, catalog number: 23595.328)

52. Trifluoroacetic acid (TFA) (Carl Roth, catalog number: 6957.1)

53. Methanol (Honeywell, catalog number: 154903)

54. HEPES (AppliChem, catalog number: A1069)

55. NaOH (Sigma-Aldrich, catalog number: 06203)

Solutions

1. Drop-out-mix, 20× (DoM) (see Recipes)

2. Galactose, 30% (see Recipes)

3. S, 5× (see Recipes)

4. S-biotin, 5× (see Recipes)

5. SGal medium (see Recipes)

6. SGal medium lacking biotin (SGal-biotin medium) (see Recipes)

7. MP1 buffer (see Recipes)

8. Sorbitol, 2.4 M (see Recipes)

9. Sorbitol, 1.2 M (see Recipes)

10. KH₂PO₄, 1 M (see Recipes)

11. K₂HPO₄, 1 M (see Recipes)

12. KPi buffer, 1 M, pH 7.4 (see Recipes)

13. MP2 buffer (see Recipes)

14. Tris, 1 M, pH 7.4 (see Recipes)

15. Tris, 20 mM (see Recipes)

16. EDTA, 0.5 M, pH 8.0 (see Recipes)

17. Homogenization buffer (HB) (see Recipes)

18. HEPES, 1 M, pH 7.4 (see Recipes)

19. SH buffer (see Recipes)

20. Biotin-phenol, 5.5 mM (see Recipes)

21. H₂O₂, 100 mM (see Recipes)

22. Ascorbic acid, 250 mM (see Recipes)

23. Sodium azide, 1% (see Recipes)

24. Trolox, 100 mM (see Recipes)

25. Quencher cocktail (see Recipes)

26. NaPi buffer, 200 mM, pH 7.4 (see Recipes)

27. NaCl, 5 M (see Recipes)

28. Sodium dodecyl sulfate (SDS), 10% (see Recipes)

29. Protease inhibitor, 10× (see Recipes)

30. Lysis buffer (RIPA) (see Recipes)

31. Urea, 8 M (see Recipes)

32. Trypsin, 0.5 μg/μL (see Recipes)

33. Dithiothreitol (DTT), 1 M (see Recipes)

34. Elution buffer I (see Recipes)

35. Chloroacetamide (CAA), 400 mM (see Recipes)

36. Elution buffer II (see Recipes)

37. Trifluoroacetic acid (TFA), 10% (see Recipes)

38. Buffer A (see Recipes)

39. Buffer B (see Recipes)

40. Buffer C (see Recipes)

Recipes

1. Drop-out-mix, 20× (DoM)

Autoclave at 120 °C for 20 min.

2. Galactose, 30%

Dissolve 30 g of galactose in up to 100 mL of ddH₂O by stirring and heating up. Autoclave at 120 °C for 20 min.

3. S, 5×

Autoclave at 120 °C for 20 min.

4. S-biotin, 5×

Autoclave at 120 °C for 20 min.

5. SGal medium

Mix reagents in an autoclaved bottle.

6. SGal-biotin medium

Autoclave at 120 °C for 20 min.

7. MP1 buffer

8. Sorbitol, 2.4 M

Dissolve 43.72 g of sorbitol in ddH₂O to a final volume of 100 mL. Autoclave at 120 °C for 20 min.

9. Sorbitol, 1.2 M

Dissolve 21.86 g of sorbitol in ddH₂O to a final volume of 100 mL. Autoclave at 120 °C for 20 min.

10. KH₂PO₄, 1 M

Dissolve 13.61 g of KH₂PO₄ in ddH₂O to a final volume of 100 mL.

11. K₂HPO₄, 1 M

Dissolve 171.8 g of K₂HPO₄ in ddH₂O to a final volume of 1 L.

12. KPi buffer, 1 M, pH 7.4

Mix 50 mL of 1 M KH₂PO₄ with ~525 mL of 1 M K₂HPO₄ to adjust to pH 7.4.

13. MP2 buffer

14. Tris, 1 M, pH 7,4

Dissolve 121.1 g of Tris(hydroxymethyl)aminomethane in ~800 mL of ddH₂O and adjust the pH to 7.4 by adding 37% HCl dropwise. Fill up to 1 L with ddH₂O.

15. Tris, 20 mM, pH 7.4

Mix 20 mL of 1 M Tris pH 7.4 with 980 mL of ddH₂O.

16. EDTA, 0.5 M, pH 8.0

Dissolve 18.6 g of EDTA disodium salt 2-hydrate in ddH₂O to a final volume of 100 mL. Adjust pH to 8.0 with NaOH.

17. Homogenization buffer (HB)

Critical: Add PMSF and BSA fresh and just before use.

18. HEPES, 1 M, pH 7.4

Dissolve 23.8 g of HEPES in ddH₂O to a final volume of 100 mL. Adjust to pH 7.4 with NaOH.

19. SH buffer

20. Biotin-phenol, 5.5 mM

Dissolve 2 mg of biotin-phenol in DMSO to a final volume of 1 mL.

21. H₂O₂, 100 mM

Dilute 5 μL of 30% H₂O₂ in 495 μL of ddH₂O.

22. Ascorbic acid, 250 mM

Dissolve 44 mg of ascorbic acid in ddH₂O to a final volume of 1 mL.

23. Sodium azide, 1%

Dissolve 100 mg of sodium azide in 9.9 mL of ddH₂O.

24. Trolox, 100 mM

Dissolve 25 mg of Trolox in ddH₂O to a final volume of 1 mL.

25. Quencher cocktail

26. NaPi buffer, 200 mM, pH 7.4

Dissolve 0.47 g of NaH₂PO₄·H₂O and 2.355 g of Na₂HPO₄ in ddH₂O to a final volume of 100 mL.

27. NaCl, 5 M

Dissolve 58.44 g of NaCl in ddH₂O to a final volume of 200 mL.

28. SDS, 10%

Dissolve 10 g of SDS in up to 100 mL of ddH₂O.

29. Protease inhibitor, 10×

Dissolve 1 tablet of protease inhibitor in 1 mL of ddH₂O.

30. RIPA

31. Urea, 8 M

Dilute 96.1 g of urea in MS-grade ddH₂O to a final volume of 200 mL.

32. Trypsin, 0.5 μg/μL

Dissolve 20 μg of trypsin (1 ampule) in 40 μL of trypsin resuspension buffer. To activate the trypsin, incubate at 30 °C for 15 min. Leftover trypsin solution can be stored at -80 °C.

33. DTT, 1 M

Dissolve 154 mg of DTT in MS-grade ddH₂O to a final volume of 1 mL

34. Elution buffer I

35. CAA, 400 mM

Dissolve 37.4 mg of CAA in MS-grade ddH₂O to a final volume of 1 mL.

36. Elution buffer II

37. TFA, 10%

Dissolve 5 mL of TFA in 45 mL of MS-grade ddH₂O.

38. Buffer A

Dissolve 50 μL of formic acid in 50 mL of MS-grade ddH₂O.

39. Buffer B

Mix 50 μL of formic acid, 40 mL of acetonitrile, and 10 mL of MS-grade ddH₂O.

40. Buffer C

Mix 100 μL of 10% TFA, 10 μL of formic acid, and 10 mL of MS-grade ddH₂O.

Laboratory supplies

1. 1.5 mL microtubes (Eppendorf, catalog number: 0030121023)

2. pH-indicator paper (Acilit, Merck, catalog number: 1.09560.0003)

3. Solid-phase extraction disk (C18 membrane) (Supelco, catalog number: 66883-U)

4. epT.I.P.S. (MS stage tips) (Eppendorf, catalog number: 022491539)

5. Erlenmeyer flasks (100, 500, 3,000 mL)

6. Pipette tips (10, 200, 1,000 μL)

7. Glass pipettes (5, 10, 20 mL)

8. Falcons (15, 50 mL)

9. Plastik pipette (5 mL)

10. Cuvettes

11. Ice

Equipment

1. Cell density meter (Fisher Scientific, model: Harvard Biochrom Ultrospec^TM 10, catalog number: 10704417)

2. High-performance centrifuge (Beckman Coulter, model: Avanti J-26 XP, catalog number: B42205)

3. 500 mL centrifugation bottles (Beckman Coulter, catalog number: 355607)

4. Dounce homogenizer plunger + cylinder (Sartorius, catalog numbers: 8530750, 8530939)

5. UV/Visible spectrophotometer (Biochrom, model: Ultrospec 2100 pro)

6. Pipette controller (Sigma-Aldrich, model: Hirschmann pipetus, catalog number: Z314951)

7. Cell disruptor (Scientific Industries, model: Disruptor genie, catalog number: D238)

8. Magnetic rack (Thermo Fisher, catalog number: 12321D)

9. Thermo shaker (Eppendorf, model: ThermoMixer C, catalog number: 5382000015)

10. Tube rotator (Stuart Scientific, model: SB1)

11. Spring-loaded syringe [18]

12. Stage tipping centrifuge (Fisher Scientific, model: Sonation, catalog number: NC2824232)

13. In-house 3D-printed frame to elute the stage tips (Figure 2) [19]

Figure 2. Model of the in-house 3D-printed frame. This model places the loaded stage tips over 200 μL reaction tubes. It enables elusion by centrifugation in a Beckman JS-5.3 rotor [19].

14. Vacuum centrifuge (Eppendorf, model: Concentrator plus, catalog number: 5305000509)

15. Scale

16. Polystyrene box

17. Dewar flask

18. Incubator

Procedure

A. Yeast cultivation

1. Inoculate a fresh colony of S. cerevisiae in 20 mL of liquid SGal medium and incubate overnight at 130 rpm and 30 °C.

Note: The experiment is performed with biological replicates. Use four different starting colonies for each strain and condition.

2. Dilute the overnight culture in 200 mL of liquid SGal medium to an optical density of 0.2 at 600 nm (OD₆₀₀) using the Ultrospec^TM cell density meter. Incubate the culture for 8 h at 130 rpm and 30°C.

3. Dilute the culture in 2 L of liquid SGal-biotin medium to an OD₆₀₀ of 0.025 and incubate overnight (18 h) at 130 rpm and 30 °C.

Note: These dilutions are based on the growth behavior of the cells. Adapt the dilutions to maintain exponential growth before starting the mitochondria isolation in the morning, as they may vary.

B. Mitochondria isolation

Note: A visual representation of the procedure can be found in [20].

1. Distribute the culture in six 500 mL centrifugation bottles to harvest cells in the exponential phase with a Beckman Coulter Avanti J-26 XP (rotor: JA10) at 2,800× g for 5 min.

2. Measure the weight of an empty bottle (W1).

3. Discard the supernatant and wash the cell pellets with ~15 mL of ddH₂O. Transfer the resuspended pellets to the previously weighed bottle and spin down at 2,800× g for 5 min.

4. Remove the supernatant and determine the wet weight (WW) of the cell pellet by weighing the bottle again (W2 - W1 = WW).

5. Resuspend the pellet in MP1 buffer (2 mL per g WW) and incubate for 10 min at 130 rpm and 30 °C.

6. Centrifuge at 2,800× g for 5 min and remove supernatant.

7. Wash the pellet in 50 mL of 1.2 M sorbitol, centrifuge at 2,800× g for 5 min, and remove the supernatant.

8. Dissolve 3 mg/g WW zymolyase in the MP2 buffer (6.7 mL/g WW) and resuspend the cell pellet with the prepared MP2 buffer. Incubate the cell solution for 60 min at 130 rpm and 30 °C.

Critical: Cool down the centrifuge to 4 °C and work only on ice from now on.

9. Centrifuge at 2,200× g for 5 min and remove supernatant.

10. Dissolve and resuspend the pellet in half of the volume of HB (13.4 mL/g WW) and transfer to a cooled Dounce homogenizer.

11. Place the Dounce homogenizer on ice and homogenize the cell solution by gently moving the plunger up and down 15 times.

12. Pour the homogenate back into the bottle and wash the homogenizer with the leftover HB.

Critical: The next step is to collect the supernatant and not discard it!

13. Centrifuge at 2,800× g for 5 min at 4 °C to spin down cell debris. Transfer the supernatant to a fresh bottle. Discard the pellet.

14. Repeat the previous step for as long as there is a visible pellet.

15. Centrifuge the supernatant at 17,500× g for 12 min and resuspend the pellet (mitochondria) in 5 mL of cold SH buffer. Use cut tips or a plastic pipette with bigger openings (see Figure 3).

Figure 3. Image of the mitochondrial pellet during mitochondrial isolation. Visible is a small mitochondrial pellet at the bottom of a 500 mL Beckmann centrifugation bottle. The pellet was obtained after centrifugation at 17,500× g for 12 min. The picture was taken before washing with SH buffer. The size of the mitochondrial pellet is the result of a 2 L starting culture.

16. Centrifuge at 17,500× g for 12 min and remove supernatant.

17. Resuspend the pellet in 300 μL of SH buffer.

18. Take an aliquot to determine protein concentration with a protein assay dye and a UV/Vis spectrometer (Bradford assay).

19. Dilute the mitochondrial suspension to a final concentration of 10 mg/mL with SH buffer.

20. Snap-freeze 50 μL aliquots in liquid nitrogen and store at -80 °C.

Pause point: Mitochondrial aliquots can be stored at -80 °C for more than one year.

C. Proximity labeling

1. Thaw 50 μL of mitochondrial suspension (10 mg/mL) on ice.

2. Add 38.2 μL of SH buffer and 1.8 μL of 5.5 mM biotin-phenol and incubate mitochondria for 30 min at 900 rpm and 30 °C.

3. Add 1 μL of 30% H₂O₂, vortex at low speed, and incubate for 5 min at 30 °C.

4. Add 100 μL of ice-cold quencher cocktail and centrifuge at 25,000× g for 10 min at 4 °C. Remove the supernatant.

5. Wash with 100 μL of quencher cocktail and centrifuge at 25,000× g for 10 min at 4 °C. Remove the supernatant.

6. Resuspend the pellet in 500 μL of ice-cold lysis buffer (RIPA).

7. Use the cell disruptor for 15 min at 4 °C to break up the mitochondrial membrane.

8. Centrifuge the lysate at 17,000× g for 10 min at 4 °C. Transfer the supernatant into a fresh tube.

D. Enrichment

1. Wash 40 μL of streptavidin beads 2× with 500 μL of RIPA buffer. Remove the supernatant with a magnetic rack.

2. Resuspend washed beads in 40 μL of RIPA buffer and add the mitochondrial lysate.

3. Incubate for 1 h tumbling at 4 °C.

4. Separate the beads on a magnetic rack and discard the supernatant.

5. Wash the beads 2× with 250 μL of RIPA buffer and 1× with 250 μL of 20 mM Tris buffer.

6. Again, add 250 μL of 20 mM Tris buffer and transfer everything to a fresh tube to remove excess detergent.

7. Add 50 μL of elution buffer I and incubate for 1 h at room temperature (RT) with tumbling.

8. Separate beads on a magnetic rack and transfer supernatant to a fresh tube.

9. Add 50 μL of elution buffer II to the beads and incubate for 30 min at RT with tumbling.

10. Separate beads on a magnetic rack, transfer supernatant, and combine with the first elution.

11. Continue the digest of the elution overnight (16 h) in the dark at 37 °C.

E. Stage tipping and MS

Note: See General note 1.

1. Acidify digested peptides by adding 10 μL of 10% TFA and spin down the sample at 13,000× g for 1 min.

2. Use 3× C18 membrane layers to purify peptides. Punch the C18 membranes with a spring-loaded syringe and transfer them to a pipette tip [18].

3. Equilibrate the tips with 100 μL of methanol and wash 2× with 100 μL of buffer A. Always spin down the volume using a stage tipping centrifuge.

4. Load acidified peptides (100 μL) onto the stage tip.

5. Wash the peptides on the C18 membrane with 100 μL of buffer A.

Pause point: The peptide-loaded tips can be stored in the fridge for some weeks.

6. Elute peptides with 40 μL of buffer B by transferring them to a 200 μL Eppendorf tube at 500× g for 5 min.

Note: We used an in-house 3D-printed frame to place the stage tips over the Eppendorf tubes (Figure 2).

7. Evaporate the sample in a vacuum centrifuge for 2 h at RT.

8. Add 9 μL of buffer C. The sample is ready for LC–MS/MS.

Validation of protocol

This protocol has been used and validated in the following research article (Figure 1):

• Flohr et al. [15]. Dysfunctional mitochondria trap proteins in the intermembrane space. EMBO J. (2025), 44: 4352–4377, DOI: https://doi.org/10.1038/s44318-025-00486-1

General notes and troubleshooting

General notes

1. Solid-phase extraction (SPE) was performed using C18 stage tips prepared in-house by a spring-loaded syringe and an available 3D-printed frame. Alternatively, commercially available C18 spin columns or 96-well disks can be used (e.g., by preomics). The design files for the 3D-printed frame are available upon request from the authors.

2. To monitor the degree of cell wall digestion with the MP2 buffer, combine 50 μL of cell solution with 2 mL of water and 2 mL of 1.2 M sorbitol. The OD₆₀₀ should be lower in water due to the eruption of cells without cell walls.

3. This method was verified in a W303 yeast strain. We assume that this method is also applicable to other commonly used yeast lab-strains (e.g., BY4741).

Troubleshooting

1. There is no or just a small mitochondrial pellet: Using 2 L of cell suspension at 1 OD₆₀₀ is normally sufficient as starting material for a decent number of mitochondria. Different conditions or mutations can change the mitochondrial volume. To fix the problem, increase the number of cells.

Acknowledgments

Conceptualization, L.S., J.M.H.; Writing—Original Draft, L.S., J.M.H.; Funding acquisition, J.M.H.; Supervision, J.M.H.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (HE2803/9-2 to J.M.H.), the European Research Council (MitoCyto 101052639 to J.M.H.), and the Research Initiative of Rheinland-Pfalz (BioComp to J.M.H.).

This protocol provides details about the methodology described recently [15], based on initial APEX labeling strategies that were pioneered by Hyun-Woo Rhee, Alice Ting, and co-workers [12].

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

This protocol exclusively uses yeast cells and does not involve any human or animal subjects.

References

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