Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells

Summary

Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM.

For complete details on the use and execution of this protocol, please refer to Kim et al.¹

Graphical abstract

Highlights

• •Protocol to assess the stalling of mitochondrial precursors in TOM in human cell lines
• •Mitochondrial enrichment and protease protection assay determine precursor localization
• •Co-immunoprecipitation demonstrates precursor arrest inside TOM

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM.

Before you begin

This protocol outlines the steps to assess the clogging of the translocase of the outer membrane (TOM) by mitochondrial precursor proteins in human cell lines. Specifically, we examine the fate of the mitochondrial precursor ornithine transcarbamylase (OTC) during mitochondrial stress. We use human embryonic kidney (HEK293T) cells that stably express OTC-V5. OTC is an essential enzyme in the urea cycle, localized to the mitochondrial matrix.² Notably, the N-terminal mitochondrial targeting signal (MTS) of OTC is cleaved upon import, allowing a separation of the uncleaved precursor from the mature form of the protein by SDS-PAGE and Western blot.^3,4 This protocol can be applied to any MTS-containing mitochondrial protein of interest and cell type.

A list of all the required materials and equipment is provided in the key resources table. Solutions are prepared according to the recipes outlined in the materials and equipment section.

Generation of stable cell lines expressing a gene of interest from an integrated lentiviral vector

Timing: 1–2 weeks

• 1.Prepare the necessary packaging vectors (pRSV-Rev; pMDLg/pRRE), envelope vector (pMD2.G), and the lentiviral transfer vector carrying the transgene of interest and antibiotic resistance marker (pLV-EF1a-IRES-Hygro).

Note: In this protocol, we fuse a V5 tag to the C-terminus of OTC in the lentiviral transfer plasmid (Figure 1A). Refer to the key resources table for additional details.

Note: This protocol describes the use of third-generation lentiviral vectors, which are comprised of four independent plasmids: the transfer vector, two packaging vectors, and the envelope vector (Figure 1B). The third-generation packaging system provides an additional layer of safety by splitting the packaging system into two separate plasmids: one encoding gag/pol and the other encoding rev. This reduces the potential for recombination events that could lead to the generation of replication-competent lentivirus.

• 2.
  Culturing and seeding HEK293FT packaging cells for transfection

  • a.
    Seed HEK293FT cells into a 100 mm tissue culture dish. Grow cells in complete DMEM media (see materials and equipment) at 5% CO₂ and 37°C.

    Note: For optimal transduction use cells passaged no more than 10 times.

  • b.
    Once the cells have reached ∼70–80% confluency, aspirate the media and gently rinse the dish with ∼3 mL PBS.
  • c.
    Incubate cells with 1 mL dissociation reagent (e.g., TrypLE Express enzyme) for 1–2 min at 37°C until the cells have detached from the surface.
  • d.
    Add 3 mL of fresh pre-warmed complete media to the dish to inactivate the dissociation reagent.
  • e.
    Transfer the cell suspension to a 15 mL tube, and pellet the cells at 400 x g for 5 min. Discard the supernatant.
  • f.
    Resuspend the cell pellet with 1 mL complete media, and count cells with Trypan Blue.
  • g.
    Seed ∼4 × 10⁶ cells into a 150 mm tissue culture dish containing 25 mL of complete media. Incubate the cells at 5% CO₂ and 37°C for 16–24 h.
• 3.
  Transfecting HEK293FT cells using calcium phosphate method

  Note: Lipid-based reagents, such as X-tremeGENE HP DNA transfection reagent, offer an alternative method for transfection.

  • a.
    Once the HEK293FT cells in the 150 mm dish have reached 70–80% confluency, remove the old media and gently replace with 25 mL fresh pre-warmed media containing 25 μM chloroquine diphosphate (1:1000 dilution). Incubate the cells at 5% CO₂ and 37°C for 5 h.

    Note: Chloroquine enhances the transfection efficiency. Refer to materials and equipment for instructions to prepare a 25 mM chloroquine diphosphate stock solution.

  • b.
    Prepare the transfection mixture in a 15 mL tube based on the volumes for each reagent listed in Table 1.
  • c.
    Slowly add 1125 μL of 2x HBS dropwise to the tube while vortexing at low speed. Incubate the transfection mixture for 30 min at 22°C–25°C.
  • d.
    Add the transfection mixture dropwise to the 150 mm tissue culture dish. Incubate the cells at 5% CO₂ and 37°C for 16–24 h.
  • e.
    12–18 h post transfection, replace media with 30 mL fresh pre-warmed complete media to remove CaCl₂.

    CRITICAL: Calcium phosphate transfection should be performed as late as possible during the day to ensure that the media is replaced the following morning post-transfection, as prolonged exposure to calcium phosphate is harmful for cell viability.

    Note: For X-tremeGENE HP transfection,

  • f.
    Prepare the transfection mixture in a 15 mL tube based on the volumes for each reagent listed in Table 2.
  • g.
    Add the transfection mixture dropwise to the 150 mm tissue culture dish. Incubate the cells at 5% CO₂ and 37°C for 16–24 h.
  • h.
    12–18 h post transfection, replace media with 30 mL fresh pre-warmed complete media.
• 4.
  Harvesting viral supernatant.

  • a.
    36–48 h post-transfection, remove the media. Gently rinse the dish with PBS, and replace with 30 mL fresh complete media.
  • b.
    After 24 h, harvest the supernatant and filter through the vacuum filtration system (0.45 μm).

Pause point: The viral particles are ready to be used. They can be stored at 4°C for 2 weeks or aliquot and store at −80°C long-term. Otherwise, proceed to step 5.

• 5.
  Lentiviral transduction and selection.

  • a.
    24 h prior to lentiviral transduction, seed 1 × 10⁶ target cells in a 100 mm dish to 50% confluency upon transduction.

    Note: Prepare a plate for uninfected control (polybrene only), empty vector virus, and virus containing the gene of interest.

  • b.
    Replace the media with 10 mL filtered virus particles, 2 mL fresh media, and 1 μg/mL polybrene.
  • c.
    24 h post-infection, remove media, wash with PBS, and replace with fresh media. If confluency is too high (∼50–60%), split the cells.
  • d.
    After 24 h, wash with PBS, remove media, and replace with media containing desired antibiotic (e.g., 200–400 μg/mL hygromycin). Observe the cells under microscope daily, and perform routine media changes every 2–3 days.

Figure 1.

Generation of cell lines stably expressing the mitochondrial protein OTC

(A) Plasmid map of pLV-OTC-V5. The pLV-EF1a-IRES-Hygro backbone vector was digested with BamHI and EcoRI restriction enzymes, and OTC-V5 was inserted by Gibson assembly. BamHI and EcoRI sites were not regenerated.

(B) Schematic workflow.

Table 1.

Volume of reagents for calcium phosphate transfection mixture

Reagent	Amount (μg)	Volume (μL)
Transfer plasmid: pLV-OTC-V5 (x μg/μL)	15	15 / x
Packaging plasmid: pRSV-Rev (y μg/μL)	4.4	4.4 / y
Packaging plasmid: pMDLg/pRRE (z μg/μL)	8.7	8.7 / z
Envelope plasmid: pMD2.G (a μg/μL)	5.3	5.3 / a
2 M CaCl2	N/A	155
ddH2O	N/A	to total volume
Total	N/A	1125

Table 2.

Volume of reagents for X-tremeGENE HP DNA transfection mixture

Reagent	Amount (μg)	Volume (μL)
Transfer plasmid: pLV-OTC-V5 (x μg/μL)	15	15 / x
Packaging plasmid: pRSV-Rev (y μg/μL)	4.4	4.4 / y
Packaging plasmid: pMDLg/pRRE (z μg/μL)	8.7	8.7 / z
Envelope plasmid: pMD2.G (a μg/μL)	5.3	5.3 / a
X-tremeGENE HP DNA transfection reagent	N/A	100
Opti-MEM I reduced serum medium	N/A	to total volume
Total	N/A	2250

Seeding cells and inducing mitochondrial stress

Timing: 2 days

This step describes seeding OTC-V5 stable cells generated in the previous step for induction of mitochondrial stress and inhibition of protein import.

Note: Mitochondrial protein import is inhibited using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and valinomycin.^5,6 These ionophores disturb the mitochondrial membrane potential and consequently inhibit protein import. However, the integrated stress response (ISR) can suppress the accumulation of precursors, as the ISR is known to be induced by various mitochondrial stressors in mammalian cells, including CCCP.^{1,6,7,8,9,10,11,12} ISR activation leads to enhanced phosphorylation of eIF2α and subsequent attenuation of global translation.^6,8,13,14 To allow the production of OTC precursors during mitochondrial stress, cells are treated with the ISR inhibitor (ISRIB) in addition to CCCP/valinomycin.

• 6.
  Seed ∼8-10 × 10⁶ HEK293T OTC-V5 stable expression cells in 150 mm tissue culture dish containing 25 mL of complete media. Incubate at 5% CO₂ and 37°C for 16–24 h.

  • a.
    For mitochondrial enrichment (described in steps 1–5), prepare two dishes: one for untreated cells, and the other for CCCP/valinomycin/ISRIB treatments.
  • b.
    For co-immunoprecipitation (described in steps 6–9), additionally seed ∼8-10 × 10⁶ HEK293T parental cells. Prepare two dishes for each cell line: one for untreated cells, and the other for CCCP/valinomycin/ISRIB treatment.

Note: Since HEK293T parental cells do not express the bait protein, OTC-V5, this will control for non-specific binding of the prey proteins to the beads or antibody.

• 7.The following day, add the following treatments according to the volumes and concentrations of the drugs listed in Table 3. Incubate at 5% CO₂ and 37°C for 16–24 h, after which the cells will be ready to be harvested for either mitochondrial enrichment or co-immunoprecipitation, as described in step-by-step method details.

Table 3.

Amounts of drugs for inducing mitochondrial protein import defects

Reagent	Stock concentration	Final concentration	Dilution	Volume (μL)
CCCP	20 mM	10 μM	1:2000	12.5
Valinomycin	1 mM	1 μM	1:1000	25
ISRIB	0.5 mM	0.5 μM	1:1000	25

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal anti-β-Tubulin (clone S11B) 1:1,000	Absolute Antibody	Cat# Ab00404–23.0
Rabbit monoclonal anti-COXIV (clone 3E11) 1:1,000	Cell Signaling Technology	Cat# 4850; RRID: AB_2085424
Rabbit monoclonal anti-TOMM20 (clone EPR15581-54) 1:1,000	Abcam	Cat# ab186735; RRID: AB_2889972
Rabbit monoclonal anti-TOMM40 (clone EPR6932(2)) 1:1,000	Abcam	Cat# ab185543, RRID: AB_3095412
Mouse monoclonal anti-V5 (clone SV5-Pk1) 1:1,000	Abcam	Cat# ab27671; RRID: AB_471093
Rat monoclonal anti-V5 (clone SV5-P-K) 1:1,000	Abcam	Cat# ab206571
Sheep anti-mouse HRP conjugated 1:10,000	Cytiva	Cat# NA931; RRID: AB_772210
Donkey anti-rabbit HRP conjugated 1:10,000	Cytiva	Cat# NA934; RRID: AB_772206
TrueBlot anti-rabbit HRP conjugated 1:10,000	Rockland	Cat# 18-8816-31; RRID: AB_2610847
Anti-mouse DyLight 800 conjugated 1:15,000	Cell Signaling Technology	Cat# 5257; RRID: AB_10693543
Anti-rabbit DyLight680 conjugated 1:15,000	Cell Signaling Technology	Cat# 5366; RRID: AB_10693812
Chemicals, peptides, and recombinant proteins
CCCP	Thermo Scientific Chemicals	Cat# 228130010
Valinomycin	Invitrogen	Cat# V1644
ISRIB	Sigma-Aldrich	Cat# SML0843
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco	Cat# 11995065
Fetal bovine serum (FBS)	Gibco	Cat# 12483020
L-glutamine	Gibco	Cat# 25030081
X-tremeGENE HP DNA transfection reagent	Roche	Cat# 6366236001
Opti-MEM I reduced serum medium	Gibco	Cat# 31985070
Penicillin-streptomycin	Gibco	Cat# 15140122
PBS	Gibco	Cat# 10010023
TrypLE Express enzyme (1x), no phenol red	Gibco	Cat# 12604013
Polybrene infection/transfection reagent	Sigma-Aldrich	Cat# TR-1003-G
Hygromycin B	Roche	Cat# 10843555001
cOmplete protease inhibitor cocktail tablets	Roche	Cat# 11836153001
Proteinase K	Sigma-Aldrich	Cat# 3115879001
PMSF	Thermo Fisher Scientific	Cat# 215740100
Dynabeads Protein G beads	Invitrogen	Cat# 10004D
5x Protein assay dye reagent concentrate	Bio-Rad	Cat# 5000006EDU
Precision Plus All Blue protein standards	Bio-Rad	Cat# 1610373
TGX Stain-Free FastCast 12% acrylamide kit	Bio-Rad	Cat# 1610185
Ammonium persulfate (APS)	Bio-Rad	Cat# 1610700
TEMED	Bio-Rad	Cat# 1610800
Critical commercial assays
Gibson Assembly Master Mix	New England Biolabs	Cat# E2611
Experimental models: Cell lines
HEK293T	ATCC	Cat# CRL-3216
HEK293FT	Thermo Fisher Scientific	Cat# R70007
Recombinant DNA
pLV-EF1a-IRES-Hygro	Hayer et al.15	Addgene plasmid #85134
pLV-OTC-V5	Kim et al.1	N/A
pRSV-Rev	Dull et al.16	Addgene plasmid #12253
pMDLg/pRRE	Dull et al.16	Addgene plasmid #12251
pMD2.G	Gift from Didier Trono	Addgene plasmid #12259
Software and algorithms
BioRender	BioRender	https://biorender.com
Others
100 × 20 mm surface tissue culture dish	Sarstedt	Cat# 83.3902
150 × 22 mm tissue culture dish	VWR	Cat# 10062-882
Falcon 15 mL conical centrifuge tube	Corning	Cat# 352095
Syringe filter, 0.2 μm pore size	VWR	Cat# 28145-501
Vacuum filtration system, 0.45 μm pore size	VWR	Cat# 10040-462
Cell scraper	Sarstedt	Cat# 83.3951
Dounce homogenizer	Wheaton	Cat #357544
5415R refrigerated centrifuge	Eppendorf	Cat# EP-5415R
Avanti J-30I high-speed centrifuge	Beckman Coulter	N/A
JA-25.50 fixed-angle rotor	Beckman Coulter	Cat# 363055
Roto-Mini rotator	Benchmark	Cat# 1159P32
Mini-PROTEAN electrophoresis cell	Bio-Rad	Cat# 1658005
Trans-Blot Turbo Transfer System	Bio-Rad	Cat# 1704271
ChemiDoc MP imaging system	Bio-Rad	Cat# 12003154

Materials and equipment

Complete DMEM media

Reagent	Final concentration	Amount
DMEM high glucose	1x	440 mL
FBS	10%	50 mL
L-glutamine	2 mM	5 mL
Penicillin/streptomycin	100 U/mL	5 mL
Total	N/A	500 mL

Keep sterile and store at 4°C until the manufacturer’s expiration date.

2x HBS, pH 7.05

Reagent	Final concentration	Amount
NaCl	280 mM	8.18 g
KCl	10 mM	0.373 g
Na2HPO4	1.5 mM	0.106 g
Dextrose	12 mM	1.08 g
HEPES, pH 7.05	50 mM	5.96 g
ddH2O	N/A	Complete to 500 mL
Total	N/A	500 mL

Adjust pH to 7.05 with NaOH. Filter sterilize using a 0.22 μm filter. Store at −20°C for up to a year.

Mitochondrial enrichment homogenate buffer

Reagent	Stock concentration	Final concentration	Amount
HEPES/KOH, pH 7.4	1 M	250 mM	125 mL
Sucrose	1 M	250 mM	125 mL
EDTA	0.5 M	1 mM	1 mL
ddH2O	N/A	N/A	249 mL
Total	N/A	N/A	500 mL

Store at 4°C for up to a year. Immediately prior to use, add protease inhibitor cocktail to a final 1x concentration (refer to the key resources table).

Immunoprecipitation lysis buffer

Reagent	Stock concentration	Final concentration	Amount
Tris, pH 7.5	1 M	50 mM	25 mL
NaCl	2.5 M	250 mM	50 mL
IGEPAL CA-630	N/A	0.5% (v/v)	2.5 mL
ddH2O	N/A	N/A	422.5 mL
Total	N/A	N/A	500 mL

Store at 22°C–25°C for up to a year. Immediately prior to use, add protease inhibitor cocktail to a final 1x concentration (refer to the key resources table).

3x SDS sample buffer

Reagent	Stock concentration	Final concentration	Amount
Tris, pH 6.8	0.5 M	187.5 mM	18.75 mL
Glycerol	N/A	30% (v/v)	15 mL
SDS	N/A	9% (v/v)	4.5 g
Bromophenol blue	N/A	0.05%	0.025 g
β-mercaptoethanol	N/A	6% (v/v)	3 mL
ddH2O	N/A	N/A	Complete to 50 mL
Total	N/A	N/A	50 mL

Store at −20°C for up to a year.

10x SDS running buffer

Reagent	Final concentration	Amount
Tris base	250 mM	90 g
Glycine	2 M	432 g
SDS	1% (w/v)	30 g
ddH2O	N/A	Complete to 3 L
Total	N/A	3 L

Store at 22°C–25°C for up to a year.

1x SDS running buffer

Reagent	Final concentration	Amount
10x SDS running buffer	1x	100 mL
ddH2O	N/A	900 mL
Total	N/A	1 L

Store at 22°C–25°C for up to a year.

1x transfer buffer

Reagent	Final concentration	Amount
5x Trans-Blot Turbo buffer	1x	200 mL
Ethanol	20% (v/v)	200 mL
ddH2O	N/A	600 mL
Total	N/A	1 L

Store at 4°C for up to 2–3 months.

10x PBS

Reagent	Final concentration	Amount
NaCl	1.37 M	80 g
KCl	27 mM	2 g
Na2HPO4	81 mM	11.5 g
KH2PO4	14.7 mM	2 g
ddH2O	N/A	Complete to 1 L
Total	N/A	1 L

Store at 22°C–25°C for up to a year.

1x PBS with Tween-20 (PBST)

Reagent	Final concentration	Amount
10x PBS	1x	100 mL
ddH2O	N/A	900 mL
Tween-20	0.1% (v/v)	1 mL
Total	N/A	1 L

Store at 22°C–25°C for up to 2–3 months.

Other solutions

Name	Reagents
10% APS	Dissolve 1 g APS in 10 mL ddH2O. Store at 4°C for 2–3 months.
CaCl2	2 M CaCl2: dissolve 29.402 g CaCl2 in 100 mL ddH2O. Filter sterilize using a 0.22 μm filter. Store at −20°C for up to a year.
CCCP	20 mM CCCP: dissolve 0.2047 g CCCP in 50 mL DMSO. Store into 50–100 μL aliquots at −20°C for up to a year.
Chloroquine diphosphate	25 mM chloroquine diphosphate; dissolve 0.129 g of chloroquine diphosphate salt into 10 mL ddH2O. Filter-sterilize through a 0.22 μm filter. Store into 50–100 μL aliquots at −20°C for up to a year.
ISRIB	0.5 mM ISRIB; dissolve 12.46 mg ISRIB in 50 mL DMSO. Store into 50–100 μL aliquots at −20°C for up to a year.
PMSF	100 mM PMSF; dissolve 0.348 g PMSF in 20 mL ethanol. Store into 50–100 μL aliquots at −20°C for up to a year.
Proteinase K	20 mg/mL proteinase K; dissolve 11.56 g proteinase K in 20 mL ddH2O. Store into 50–100 μL aliquots at −20°C for up to a year.
5% skimmed milk/1x PBST	Add 5 g skimmed milk in 100 mL 1x PBST Store at 4°C for up to 1 week.
Valinomycin	1 mM valinomycin; dissolve 55.76 mg valinomycin in 50 mL ethanol. Store into 50–100 μL aliquots at −20°C for up to a year.

Step-by-step method details

Mitochondrial enrichment and protease protection assay

Timing: 2 h

The following steps describe the fractionation of HEK293T cells in order to assess the cellular localization of mitochondrial precursor accumulation upon impaired protein import (Figure 2).

Note: We use this technique to detect mitochondrial precursors in cytosolic and mitochondrial enriched-fractions. Moreover, the detection of precursors within the mitochondrial fraction can indicate defects in either import into the organelle or MTS cleavage in the matrix. To distinguish between these possibilities, we use a protease protection assay whereby mitochondrial-enriched fractions from stressed OTC-V5-expressing cells are subjected to proteinase K treatment. As proteinase K can only process proteins that are exposed to the surface of the mitochondria, fully imported proteins are not affected by this treatment (Figure 2).

CRITICAL: All steps should be performed on ice to prevent proteolysis. Pre-chill all buffers, tubes, and the Dounce homogenizer on ice.

• 1.
  Harvest cells.

  • a.
    Prepare the homogenate buffer by dissolving 1 tablet of cOmplete protease inhibitor cocktail in 10 mL of homogenate buffer (see materials and equipment for recipe).
  • b.
    Remove media from 150 mm dish with at least 80% density of cells and wash the cells with PBS. Aspirate the PBS.
  • c.
    Add 5 mL homogenate buffer with protease inhibitors to each culture dish.
  • d.
    Detach the cells from the dish with a cell scraper.
  • e.
    Transfer the cell suspension to a fresh 15 mL tube, and keep on ice.
• 2.
  Homogenize the cells.

  • a.
    Using a glass Dounce homogenizer, homogenize the cell suspension by applying 20–25 strokes with the homogenizer’s tight-fitting pestle (B pestle).
  • b.
    Transfer the homogenate to a clean centrifuge tube.
  • c.
    Reserve a small portion of the total cell homogenate (approximately 100–150 μL).
• 3.
  Low-speed centrifugation.

  • a.
    Centrifuge the homogenate at 800 x g for 5 min at 4°C.
  • b.
    Transfer the supernatant to a clean centrifuge tube.

Note: The low-speed centrifugation pellet contains unbroken cells, cell debris, and intact nuclei. The supernatant contains the cytosol and the organelles.

• 4.
  High-speed centrifugation.

  • a.
    Centrifuge the supernatant at 10,000 x g for 10 min at 4°C.
  • b.
    Transfer 1 mL of the supernatant to a clean 1.5 mL centrifuge tube.
  • c.
    Gently resuspend the pellet in 500 μL of homogenate buffer.

    Note: High-speed centrifugation results in a pellet that contains crude mitochondria (mitochondrial-enriched fraction). The supernatant contains the cytosol and other organelles.

  • d.
    Centrifuge again at 10,000 x g for 10 min.
  • e.
    Gently resuspend the pellet in 100 μL homogenate buffer.
• 5.
  Protease protection assay and sample preparation.

  • a.
    Estimate the protein concentration of the mitochondrial and cytosolic fractions, as well as the total cell homogenate, by Bio-Rad Protein Assay according to the manufacturer’s protocol.

    • i.
      Transfer ∼10–20 μg of the total cell homogenate and cytosolic fraction to a clean 1.5 mL Eppendorf tube.
    • ii.
      Add 3x SDS sample buffer.
    • iii.
      Boil at 95°C for 5 min.
  • b.
    To ∼10–20 μg of the mitochondrial fraction, add proteinase K to a final concentration of 0.25–1 mg/mL. Include a control sample without proteinase K. Incubate for 30 min at 37°C.
  • c.
    Add PMSF to a final concentration of 4 mM and incubate on ice for 15 min to stop the proteinase activity.
  • d.
    Add 3x SDS sample buffer. Boil the samples at 95°C for 5 min.

Pause point: If not immediately performing SDS-PAGE, the total cell homogenate, cytosolic and mitochondrial fractions that have already been boiled at 95°C with SDS sample buffer can be stored at −20°C.

Figure 2.

Schematic workflow of mitochondrial enrichment and protease protection assay

Co-immunoprecipitation

Timing: 5–6 h

Mitochondrial enrichment and protease protection assay determine whether precursors accumulate at the mitochondrial surface when protein import is impaired. To further examine stalling inside TOM, we immunoprecipitated a mitochondrial precursor, such as OTC, to test its association with TOM subunits. The following steps describe the protocol for immunoprecipitating OTC-V5 from both untreated cells and cells with mitochondrial stress using the Dynabeads Protein G Immunoprecipitation Kit (Figure 3).

Note: Using magnetic bead-V5 antibody conjugate (for example: Cell Signaling Cat# 31628) could provide an alternative to the Dynabeads Protein G Immunoprecipitation Kit. When using the antibody-bead conjugate, skip steps 6 and 8a-d. Instead, directly add 20 μL conjugate to the lysate remaining after step 7g.

• 6.
  Bind antibody to Dynabeads.

  • a.
    Resuspend Dynabeads in the vial (vortex for at least 30 s).
  • b.
    For each reaction, transfer 50 μL (1.5 mg) Dynabeads to a clean 1.5 mL centrifuge tube.
  • c.
    Place the tubes on a magnet to adhere the beads to the side of the tube and discard the supernatants.
  • d.
    Add 3–5 μg of V5 antibodies diluted in 200 μL ice-cold PBS to each tube containing Dynabeads.
  • e.
    Incubate with rotation for 30 min at 22°C–25°C.
• 7.
  Lyse cells.

  • a.
    Prepare sufficient immunoprecipitation lysis buffer by adding 1 tablet of cOmplete protease inhibitor cocktail per 10 mL of lysis buffer (see materials and equipment for recipe).
  • b.
    Remove media from 150 mm dish with at least 80% density of cells and wash the cells with PBS. Aspirate the PBS.
  • c.
    Add 1.5 mL lysis buffer with protease inhibitors to each culture dish to lyse the cells.
  • d.
    Use a cell scraper to collect the lysate from the dish and transfer it to a clean 1.5 mL centrifuge tube. Incubate on ice for 10 min.
  • e.
    Centrifuge at 16,000 x g for 10 min at 4°C.
  • f.
    Transfer the supernatant to a new 1.5 mL centrifuge tube and keep on ice.
  • g.
    Transfer approximately 2% of the lysate to a new tube (∼30 μL). Add 3x SDS sample buffer and incubate at 95°C for 5 min. This sample is used as the input fraction for SDS-PAGE.
• 8.
  Immunoprecipitate target antigen.

  • a.
    Place the tube containing the bead-antibody complex (from step 6e) on the magnet to remove the supernatant.
  • b.
    Gently resuspend the bead-antibody complex with 200 μL ice-cold PBS.
  • c.
    Place the tube back onto the magnet and discard the supernatant.
  • d.
    Transfer the remaining lysate (from step 7f) to the bead-antibody complex and resuspend by gentle pipetting.
  • e.
    Incubate with rotation for 2 h at 4°C to allow the target antigen to bind to the bead-antibody complex.
  • f.
    Once the incubation is complete, place the tube back onto the magnet and discard the supernatant.
  • g.
    Wash: gently resuspend the bead-antibody complex with 200 μL ice-cold PBS. Place the tube back onto the magnet and discard the supernatant.
  • h.
    Repeat step 8g to a total of 3 washes.
  • i.
    Final wash: resuspend the bead-antibody-antigen complex with 100 μL ice-cold 1x PBS and transfer the suspension to a clean 1.5 mL centrifuge tube. This is recommended to avoid co-elution of proteins bound to the tube wall.
• 9.
  Elute target antigen.

  • a.
    Place the tube from step 8i onto the magnet and remove the supernatant.
  • b.
    Add 30 μL of 1x SDS sample buffer, and carefully resuspend the bead complex.
  • c.
    Boil the samples at 95°C for 5 min.

Pause point: The input (step 7g) and immunoprecipitation (step 9c) samples that have boiled at 95°C with SDS sample buffer can be stored at −20°C.

Figure 3.

Schematic workflow of co-immunoprecipitation

SDS-PAGE and western blot analysis

Timing: 1–2 days

The following steps describe the protocol for SDS-PAGE and subsequent Western blot analysis for the detection of mitochondrial precursors, such as OTC, as well as their interacting partners.

Note: Most of the mitochondrial proteins, including OTC, contain an N-terminal MTS that is cleaved upon import.^3,4 This allows for separation of the OTC precursor (upper band; ∼40 kDa) from its mature form (lower band; ∼37 kDa) when analyzing the cytosolic and mitochondrial-enriched fractions (as well as the total cell homogenate) collected in steps 1–5. In addition, a mitochondrial marker (e.g. COXIV) and outer mitochondrial membrane marker (e.g. TOMM40) will be detected. The input and immunoprecipitation samples obtained in steps 6–9 will be analyzed on a separate gel and blotted for OTC precursors, along with subunits of the TOM complex—TOMM20 and TOMM40. This will determine whether TOM co-immunoprecipitates with OTC precursors under stress conditions. Here, we utilize TGX Stain-Free FastCast acrylamide solutions (Bio-Rad) for gel electrophoresis and Trans-Blot Turbo Transfer System (Bio-Rad) for protein transfer, but alternative electrophoresis and transfer methods should also be acceptable.

• 10.
  SDS-PAGE

  • a.
    Cast a 1.5 mm acrylamide gel using the TGX Stain-Free FastCast 12% acrylamide solutions according to the manufacturer’s protocol.
  • b.
    Assemble the electrophoresis apparatus. Fill the chambers with 1x SDS running buffer (see materials and equipment for recipe).
  • c.
    Load 5–7 μL of Precision Plus All Blue Protein Ladder (Bio-Rad). Then, load the samples onto subsequent wells of the gel. For empty wells, load equal volume of 1x SDS sample buffer. Run at 100–140 V.

Note: To ensure optimal separation of the precursor and mature bands of OTC, run the gel at 100–140 V right before the 37 kDa marker runs off the end of the gel. Voltage can be increased but a higher running speed may affect the quality of the separation.

• 11.
  Protein transfer.

  • a.
    Pre-wet and equilibrate one nitrocellulose membrane and two transfer stacks per gel in transfer buffer (using the Bio-Rad Trans-Blot Turbo RTA Midi Transfer Kit).
  • b.
    Assemble the “transfer sandwich” by placing one transfer stack in the bottom of the Trans-Blot Turbo cassette. Carefully place the nitrocellulose membrane on the transfer stack.
  • c.
    Turn off the electrophoresis unit, and carefully separate the two glass plates to transfer the gel onto the nitrocellulose membrane.

    Note: To visualize total protein bands on the gel, first place the gel onto the ChemiDoc MP imaging system stage. Select the “Stain-Free” option under the “Protein Gels” category. Allow 45 seconds for stain-free gel activation and image capture.

  • d.
    Add the last transfer stack on the top. Gently roll out to remove bubbles and excess transfer buffer.
  • e.
    Close and lock the cassette lid.
  • f.
    Insert the cassette into the Trans-Blot Turbo instrument. Navigate the control panel by first selecting “List”, then “Bio-Rad”, and followed by either “1 mini gel” or “2 mini or 1 midi gel”. Scroll down until “1.5 mm gel” is selected, and press “Run”.
  • g.
    Once the transfer is complete, carefully remove the membrane from the cassette.

    Note: To assess the efficiency of protein transfer, place the membrane onto the ChemiDoc stage. Select the “Stain-Free” option under the “Blots” category to capture the image.

  • h.
    Transfer the membrane to a container containing blocking buffer (5% skimmed milk powder in 1x PBST). Incubate for 30 min at 22°C–25°C on a rocking shaker.
• 12.
  Antibody incubation and image acquisition

  • a.
    Probe the membrane with the appropriate primary antibody (diluted in 1% BSA/1% skimmed milk/0.05% sodium azide) on a rocking shaker for 2 h at 22°C–25°C or 16–24 h at 4°C. Refer to the key resources table for antibody dilutions.
  • b.
    Wash membranes in 1x PBST three times for 5 min.
  • c.
    Incubate membranes with the appropriate secondary antibody (diluted in 5% skimmed milk/1x PBST) for 30 min. Refer to the key resources table for antibody dilutions.
  • d.
    Wash membranes in 1x PBST three times for 5 min.
  • e.
    Detect signals using the ChemiDoc MP imaging system.

Expected outcomes

We expect to detect accumulation of mitochondrial precursor proteins in cells treated with CCCP/valinomycin/ISRIB. Cellular fractionation is expected to reveal whether a mitochondrial precursor of interest accumulates in the mitochondrial or cytosolic enriched fraction, while the protease protection assay is expected to test whether the precursor is fully imported. SDS-PAGE and Western blot analyses should allow a clear separation of the precursor and mature forms of precursors in each fraction (indicated by the presence of an upper and lower band, respectively).

In the case of OTC, we expect the OTC-V5 precursor to be detected in the mitochondrial enriched fraction from OTC-V5 stably expressing cells (Figure 4A). In contrast, almost no OTC-V5 should be detected in the cytosolic fraction (Figure 4A). Moreover, the precursor, but not mature form, of OTC-V5 should exhibit sensitivity to proteinase K (Figure 4B). Since OTC-V5 is detected using a C-terminal V5 tag, this suggests that at least the C terminus of the OTC precursor resides at the mitochondrial surface, facing the cytosol.

Figure 4.

Expected outcomes of OTC precursor accumulation site upon mitochondrial stress

Representative Western blots of mitochondrial enrichment (A and B) and co-immunoprecipitation (C).

Figure reprinted and adapted with permission from Kim et al., 2024.¹

Immunoprecipitation of OTC-V5 from cells treated with CCCP/valinomycin/ISRIB is expected to pull down two components of the TOM complex: the import receptor, TOMM20, and the channel-forming subunit, TOMM40 (Figure 4C). This association can also occur under non-stressed conditions to a lesser degree, which presumably represents the transient interaction of OTC with the import machinery while it is translocated into the mitochondria. Overall, this result suggests that, upon inefficient import into the mitochondria, OTC precursor can clog the import channels in human cells.

Limitations

The provided protocol specifically examines the fate of OTC when protein import into the mitochondria is impaired. OTC is an ideal substrate because it allows for a clear separation between its precursor and mature forms, which can be effectively detected by Western blot analysis. However, when expanding this analysis to other mitochondrial proteins beyond OTC, it is important to consider that some mitochondrial precursors may present detection challenges due to smaller MTS or reduced precursor stability. Online prediction tools such as MitoFates and MTSviewer can be used to assess the size of the MTS and the location of the cleavage site.^17,18

Troubleshooting

Problem 1

The OTC precursor band cannot be detected by Western blot (in step 10 of ‘SDS-PAGE and Western blot analysis’).

Potential solution

• •Avoid growing the cells to 100% confluency before drug treatment. It is recommended to seed the cells so that they achieve ∼50–60% confluency by the following day.
• •Adjust SDS-PAGE conditions by increasing acrylamide percentage of the gel or running the gel at a lower voltage to allow for better separation.

Problem 2

The mitochondrial pellet is not visible after enrichment (in steps 1 and 2 of ‘mitochondrial enrichment and protease protection assay’).

Potential solution

• •Increase the starting number of cells.
• •Ensure proper homogenization. Insufficient homogenization can leave cells intact, resulting in low mitochondrial yield.

Problem 3

Proteinase K digestion of mitochondrial fraction indicates that the mitochondria are not intact and are permeable for the protease, as evidenced by the degradation of a matrix marker (in steps 4 and 5 of ‘mitochondrial enrichment and protease protection assay’).

Potential solution

• •Gently resuspend the mitochondrial pellet to maintain the structural integrity of the mitochondria, as excessive or vigorous pipetting can cause the organelle to rupture, leading to the release of its contents.
• •Resuspending with a larger pipette tip can help reduce the risk of rupturing the mitochondrial membrane.
• •Decrease the concentration of proteinase K. We recommend titrating the proteinase K concentration to identify the optimal range for effective digestion.

Problem 4

Proteins in the cell lysate bind non-specifically to the beads themselves or the antibodies attached to the beads (in steps 6–8 of ‘co-immunoprecipitation’).

Potential solution

• •Include a pre-clearing step by incubating the lysate with beads without the antibody for 30 min at 4°C.
• •Block the beads with 0.2% bovine serum albumin (BSA) in 1x PBST for 30 min during the antibody-bead binding step. After blocking, wash the beads to remove excess BSA and incubate with the cell lysate containing a final concentration of 0.2% BSA.
• •Increase the number of washing steps.

Problem 5

The antibody used for immunoprecipitation is detected in the Western blot analysis. This problem is common when using a secondary antibody against the species of the immunoprecipitation antibody (e.g., anti-mouse or anti-rabbit). The secondary antibody can bind to both the IgG heavy (∼50 kDa) and light (∼25 kDa) chain of the immunoprecipitation antibody. This results in these bands appearing on the blot, which may hinder the detection of the protein of interest, particularly if its molecular weight is similar to that of the antibody chains (in step 12 of ‘SDS-PAGE and Western blot analysis’).

Potential solution

• •Consider using a primary antibody from a different species than the one used for detection. This will prevent the secondary antibody from binding to the IP antibody.
• •Alternatively, use a secondary antibody that only recognizes primary antibodies in their native, non-reduced states (e.g., TrueBlot secondary antibody).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hilla Weidberg (hilla.weidberg@ubc.ca).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, John Kim (john.kim@ubc.ca).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

No data or code was generated in this study.

Acknowledgments

The work was supported by the Canadian Institutes of Health Research (grant PJT-180426 to H.W.) and Michael Smith Health Research BC (SCH-2021-1524 to H.W.). J.K. received funding from the Canadian Institutes of Health Research (Canada Graduate Scholarship—Master’s award), British Columbia Graduate Scholarship, and the Korean Canadian Scholarship Foundation. The graphical abstract was created with BioRender.

Author contributions

J.K. and H.W. contributed to writing and editing the manuscript.

Declaration of interests

The authors declare no competing interests.