Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Summary

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Abstract

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Introduction

Mitochondria are essential organelles of eukaryotic cells that play crucial roles in bioenergetics, cellular metabolism, and signaling pathways¹. To properly execute these tasks, mitochondria rely on a unique set of proteins and lipids responsible for their structure and function. The budding yeast Saccharomyces cerevisiae has been widely used as a model system for investigations on mitochondrial processes, as well as for other organelles². The mitochondrial genome codes for only eight proteins in yeast; the vast majority of mitochondrial proteins (~99%) are encoded by nuclear genes, which are translated on cytosolic ribosomes, and subsequently imported into their correct submitochondrial compartments by sophisticated protein import machineries^3,4,5. Thus, mitochondrial biogenesis depends on the coordinated expression of both the nuclear and mitochondrial genomes^6,7. Genetic mutations causing defects in mitochondrial biogenesis are associated with human diseases^8,9,10.

In the past two decades, high-throughput proteomic studies targeting highly-purified mitochondria resulted in a comprehensive characterization of yeast mitochondrial proteome, which has been estimated to be composed of at least 900 proteins^11,12,13,14. Although these studies provided valuable information, the suborganellar localization of each protein in the four mitochondrial subcompartments, namely, the outer membrane (OM), intermembrane space (IMS), inner membrane (IM), and matrix, is still required. This question was partially addressed with proteomic-wide studies of the two smaller mitochondrial subcompartments (OM and IMS)^15,16. More recently, Vögtle and collaborators made a major step forward by generating a high-quality global map of submitochondrial protein distribution in yeast. Using an integrated approach combining SILAC-based quantitative mass spectrometry, different submitochondrial fractionation protocols, and the data set from the OM and IMS proteomes, the authors assigned 818 proteins into the four mitochondrial subcompartments¹³.

Despite the advances achieved by these high-throughput proteomic studies, our knowledge about the submitochondrial proteome composition is far from being complete. Indeed, among 986 proteins reported by Vögtle and collaborators as being localized into yeast mitochondria, 168 could not be assigned in any of the four submitochondrial compartments¹³. Moreover, the authors did not provide information about the membrane topology of proteins that were predicted to be peripherally attached to the periphery of mitochondrial membranes. For example, it is not possible to know if a protein that was assigned as peripherally attached to the inner membrane is facing the matrix or the intermembrane space. Apart from these missing data from the proteome-wide studies, there are conflicting information about the suborganellar localization of a significant number of mitochondrial proteins. One example is the protease Prd1, which has been assigned as an intermembrane space protein in the common databases such as Saccharomyces Genome Database (SGD) and Uniprot. Surprisingly, using a subfractionation protocol similar to that described here, Vögtle and collaborators clearly showed that Prd1 is a genuine matrix protein¹³. As mentioned above, the submitochondrial localization of many mitochondrial proteins needs to be elucidated or reevaluated. Here, we provide a simple and reliable protocol to determine the suborganellar localization of yeast mitochondrial proteins. This protocol was developed and optimized by various research groups and has been routinely used to determine the submitochondrial localization, as well as the membrane topology of many mitochondrial proteins.

Protocol

1. Growth of yeast cells

1. Isolate single colonies of the strain of interest by streaking a small portion of the cells from a -80 °C glycerol stock onto a YPD (1% yeast extract, 2% peptone, 2% glucose) agar plate. Incubate the plate at 30 °C for 2-3 days.
   NOTE: The S. cerevisiae strain used in this protocol is derived from BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0). With the exception of the auxotrophic marker genes, this strain does not contain any deleted gene and does not carry any plasmid. Thus, it can be successfully cultivated in a rich medium, stimulating vigorous cell growth. When working with strains transformed with plasmids, use the appropriate minimal medium for plasmid selection.
2. Prepare a starter culture by inoculating 2-3 individual colonies from YPD agar plate in 10-20 mL of YPGal medium (1% yeast extract, 2% peptone, 2% galactose) in a 100 mL Erlenmeyer flask. Incubate at 30 °C for 24 h with vigorous shaking.
   NOTE: The choice of the growth medium depends on the yeast strain used in the protocol. Both YPD and YPGal contain fermentable carbon sources, which allow the growth of strains that do not perform mitochondrial respiration. However, since glucose represses the expression of many mitochondrial genes, it is not recommended to use this carbon source since it will produce lower amounts of mitochondria. When working with respiratory competent strains that can respire, it is also possible to use carbon sources such as glycerol and ethanol in an attempt to obtain a higher yield of mitochondria.
3. Dilute the starter culture into 1 L of fresh YPGal medium to an OD₆₀₀ less than 0.1. Cultivate the cells at 30 °C with vigorous shaking until OD₆₀₀ reaches 1-1.5.
   ​NOTE: Determine the growth rate (doubling time) for each yeast strain before performing the experiment. This will provide an accurate estimate of the time of incubation required for the culture to reach an OD₆₀₀ of 1-1.5.

2. Isolation of highly purified mitochondria

NOTE: This protocol is adapted from¹⁷, with minor modifications.

1. Harvest the cells by centrifugation at 3,000 x g for 5 min at room temperature.
2. Wash the cells with distilled water and collect them by centrifugation at 3,000 x g for 5 min at room temperature.
3. Determine the wet weight of the cells.
   NOTE: The easiest way to measure the weight of the cell pellet from step 2.2 is to determine the weight of the empty centrifuge tube just before the collection of the cells. After centrifugation, discard the supernatant and measure the weight of the same tube with the cells. The weight of the cells is the difference between the two measurements.
4. Resuspend the cells in DTT buffer (2 mL per 1 g of cells) using a Pasteur pipette or P5000 tip. See Table 1 for DDT buffer composition.
5. Incubate the cells at 30 °C for 20 min with gentle shaking (~70 rpm).
6. Centrifuge at 3,000 x g for 5 min at room temperature to pellet the cells.
7. Wash the cells with Zymolyase buffer without the enzyme (about 7 mL per 1 g of cells).
   ​See Table 1 for Zymolyase buffer composition.
8. Centrifuge at 3,000 x g for 5 min at room temperature to pellet the cells.
9. Resuspend the cells in the buffer without Zymolyase (7 mL per 1 g of cells).
10. Transfer the cell suspension to a 250 mL Erlenmeyer flask and add Zymolyase-20T (3 mg per g wet weight).
11. Incubate the cells at 30 °C for 30-40 min with gentle shaking (~70 rpm). Check the efficiency of this process by comparing the turbidity of the cell suspension before and after Zymolyase treatment.
    NOTE: In this step, the cells will be converted into spheroplasts due to cell-wall digestion by Zymolyase.
    1. For this, add 50 µL of each cell suspension to separate glass tubes containing 2 mL of water. After mixing vigorously, the turbidity of the cell suspension treated with Zymolyase should rapidly decrease due to the osmotic disruption of spheroplasts. On the other hand, the turbidity of the non-treated cell suspension should remain unchanged.
       NOTE: The effects of turbidity can also be monitored by simple visual inspection or by measuring the OD₆₀₀ of both samples. In the second case, the OD₆₀₀ of the Zymolyase treated sample should be 10%-20% of the non-treated sample. An alternate method involves counting the cells in both samples by using light microscopy.
    2. If the yield of spheroplasts formation is low, add more Zymolyase and incubate for a further 15 min interval.
12. Harvest the spheroplasts by centrifugation at 2500 x g for 5 min at 4 °C.
    NOTE: All the further steps should be carried out fast and on ice or at 4 °C to avoid protein degradation by hydrolytic enzymes.
13. Wash the spheroplasts twice with ice-cold homogenization buffer (about 6.5 mL per 1 g of cells) and pellet by centrifuging at 2,500 x g for 5 min at 4 °C. See Table 1 for homogenization buffer composition.
14. Resuspend the spheroplasts in ice-cold homogenization buffer (6.5 mL per 1 g of cells) and transfer it to a pre-chilled glass Dounce homogenizer. Use a large glass homogenizer of approximately 30 mL.
15. Homogenize the spheroplasts with 15 strokes using a pestle.
    NOTE: The number of strokes should be adjusted depending on the pestle fitting. For tight pestle, 15 strokes are sufficient. On the other hand, if using a loose pestle, it is recommended to perform up to 25 strokes.
16. Transfer the homogenate to a 50 mL centrifuge tube and add 1 volume of ice-cold homogenization buffer.
17. Centrifuge the homogenate at low speed, 1,500 x g for 5 min at 4 °C to pellet nuclei, cell debris, and unbroken cells.
18. Transfer the supernatant to a new 50 mL centrifuge tube using a Pasteur pipette or P5000 tip taking care to avoid disrupting the pellet.
19. Centrifuge at 4,000 x g for 5 min at 4 °C.
20. Transfer the supernatant to a high-speed centrifuge tube, and centrifuge at 12,000 x g for 15 min at 4 °C to pellet the crude mitochondria fraction.
21. Discard the supernatant and gently wash the crude mitochondrial pellet in 20-30 mL ice-cold homogenization buffer by gentle pipetting using a P5000 tip.
22. Transfer the suspension to a 50 mL centrifuge tube and centrifuge at 4,000 x g for 5 min at 4 °C to pellet the remaining cell debris.
23. Transfer the supernatant to a high-speed centrifuge tube, and centrifuge at 12,000 x g for 15 min at 4 °C to pellet the crude mitochondria fraction.
24. Discard the supernatant and gently resuspend the crude mitochondrial pellet in a small volume (typically 1000 µL) of ice-cold SEM buffer by gentle pipetting using a P1000 tip. See Table 1 for SEM buffer composition.
    NOTE: Although this crude mitochondrial fraction can be used directly in some applications such as in organello protein import assays, it contains substantial amounts of other cellular components. These contaminations might lead to misinterpretations of the results when determining the submitochondrial localization of a protein. Therefore, further purification steps are required to get highly purified mitochondrial preparation, as described below.
25. Prepare sucrose solutions in the EM buffer at concentrations of 60%, 32%, 23%, and 15% (w/v). These solutions are stable for up to 1 month at 4 °C. See Table 1 for EM buffer composition.
26. Prepare a 4-step sucrose gradient in an ultracentrifuge tube as follows: Place 1.5 mL of 60% (w/v) sucrose onto the bottom of the centrifuge tube. Next, pipette carefully stepwise: 4 mL of 32%, 1.5 mL of 23%, and 1.5 mL of 15% sucrose (w/v). Take care to avoid disrupting the phases.
27. Carefully load the crude mitochondrial fraction on top of the sucrose gradient.
28. Centrifuge for 1 h at 134,000 x g at 4 °C in a swinging bucket rotor.
29. Carefully keep removing the sucrose solution until the highly purified mitochondria fraction is reached which is represented by a brown band at the 60%/32% sucrose interface.
30. Recover the purified mitochondria using a P1000 cut tip and place it into a pre-chilled high-speed centrifuge tube.
31. Dilute the recovered mitochondria with 5-10 volumes of ice-cold SEM buffer.
32. Centrifuge for 30 min at 12,000 x g at 4 °C.
33. Resuspend the pure mitochondria in 500 µL of ice-cold SEM buffer by gentle pipetting using a P1000 cut tip.
34. Determine the protein concentration of the highly purified mitochondrial preparation using the Bradford procedure following the manufacturer's instructions. Adjust the protein concentration to 10 mg of protein/mL with ice-cold SEM buffer.
    ​NOTE: For the submitochondrial fractionation protocol described below, it is recommended to use freshly prepared mitochondria. However, Vögtle and collaborators performed a detailed quality control analysis of the mitochondrial intactness and showed that frozen organelles can also be used in this protocol¹³. For this, make aliquots of 40 µL and freeze them in liquid nitrogen. Store at -80 °C.

3. Submitochondrial fractionation protocol

NOTE: This protocol is adapted from reference¹⁸ and is composed of two steps: (1) hypotonic swelling in the presence or absence of proteinase K, and (2) sonication followed by carbonate extraction. Perform all the steps of both the protocols on ice or at 4 °C to avoid protein degradation.

1. Hypotonic swelling in the presence of proteinase K
   1. Transfer 40 µL of highly purified mitochondria at 10 mg/mL (400 µg) into four 1.5 mL pre-chilled labeled microcentrifuge tubes.
   2. Add 360 µL of SEM buffer in tubes 1 and 2.
   3. Add 360 µL of EM buffer in tubes 3 and 4.
   4. Add 4 µL of proteinase K (10 mg/mL) in tubes 2 and 4. Use the pipetting scheme listed in Table 2 to avoid mistakes.
      NOTE: Prepare a 10 mg/mL solution of proteinase K in water immediately before use. The final concentration of proteinase K in the experiment should be around 50-100 µg/mL. Please see the Discussion section for further details.
   5. Mix all the tubes gently and incubate on ice for 30 min with occasional mixing.
   6. Add 4 µL of 200 mM PMSF to all the four tubes to stop proteinase K activity.
      CAUTION: PMSF is highly toxic. Wear gloves when working with solutions containing PMSF.
   7. Centrifuge at 20,000 x g for 30 min at 4 °C.
   8. Collect the supernatant and transfer it to a new 1.5 mL pre-chilled labeled microcentrifuge tube. Take care to avoid disrupting the pellet.
      NOTE: The pellet can be directly resuspended in the sample buffer for further SDS-PAGE and western blot analysis. However, traces of proteinase K can remain active even after PMSF treatment and eventually can digest some proteins after the pellet has been dissolved in the SDS-containing sample buffer. To avoid this problem, proteinase K could be completely inactivated by treatment of the samples with trichloroacetic acid (TCA) as described below.
      CAUTION: TCA is highly toxic. Wear gloves when working with solutions containing TCA.
   9. Resuspend the pellet from step 3.1.8 in 400 µL of ice-cold SEM buffer.
   10. Precipitate the supernatant (from step 3.1.8) and the resuspended pellet (from step 3.1.9) with TCA to a final concentration of 10% (w/v).
   11. Incubate all the tubes on ice for 10 min.
   12. Centrifuge the TCA-treated samples for 10 min at 12,000 x g at 4 °C.
   13. Remove the supernatant and resuspend the pellet in 200 µL of sample buffer.
       NOTE: It is possible that the bromophenol blue pH indicator turns yellow because of the acid treatment. If this happens, add small aliquots (1-5 µL) of 1 M Tris base until it turns blue.
   14. Add 4 µL of 200 mM PMSF to all the tubes.
   15. Store all the samples at -80 °C until further analysis by SDS-PAGE and western blot.
2. Sonication and carbonate extraction
   NOTE: In this protocol, it is not necessary to use freshly prepared mitochondria once the sonication causes the rupture of the mitochondrial membranes.
   1. Transfer 200 µL of highly purified mitochondria at 10 mg/mL (2 mg protein) into a 1.5 mL pre-chilled microcentrifuge tube.
   2. Dilute mitochondria one-fold with ice-cold SEM buffer.
   3. Sonicate mitochondria for 3 x 30 s on ice. Use a sonicator compatible for small volumes.
   4. Centrifuge the sample for 30 min at 100,000 x g at 4 °C.
   5. Collect the supernatant and transfer it to a new 1.5 mL pre-chilled microcentrifuge tube. Keep it on ice. This sample will be named soluble protein fraction (S).
   6. Resuspend the pellet from step 3.2.4 in 400 µL of ice-cold SEM buffer.
   7. Take 100 µL of the resuspended pellet from step 3.2.6 and transfer it to a new 1.5 mL pre-chilled microcentrifuge tube. Keep it on ice. This sample will be named submitochondrial particles fraction (SMP).
   8. Dilute the remaining 300 µL from step 3.2.6 one-fold with freshly prepared 200 mM sodium carbonate.
   9. Incubate the sample from step 3.2.8 on ice for 30 min.
   10. Centrifuge the sample for 30 min at 100,000 x g at 4 °C.
   11. Collect the supernatant and transfer it to a new 1.5 mL pre-chilled microcentrifuge tube. Keep it on ice. This sample will be named carbonate supernatant fraction (CS).
   12. Resuspend the pellet from step 3.2.10 in 400 µL of ice-cold SEM buffer. This sample will be named carbonate precipitated fraction (CP).
   13. Precipitate all the samples (S, SMP, CS, and CP) with TCA to a final concentration of 10% (w/v).
   14. Incubate all the tubes on ice for 10 min.
   15. Centrifuge the TCA-treated samples for 10 min at 12,000 x g at 4 °C.
   16. Remove the supernatant and resuspend each pellet in the sample buffer. If the sample buffer becomes yellow, add small aliquots (1-5 µL) of 1 M Tris base until it turns blue.
   17. Add 1 µL of 200 mM PMSF to all the tubes.
   18. Store all the samples at -80 °C until further analysis by SDS-PAGE and western blot.

Results

The success of submitochondrial fractionation protocol depends on obtaining highly purified intact mitochondria. For this, it is essential that during the yeast cell lysis, the intactness of the organelles remains almost totally preserved. This is achieved by using a cell lysis protocol that combines the enzymatic digestion of the cell wall followed by physical disruption of the plasma membrane by using a Dounce homogenizer. The mitochondrial contents are then collected by differential centrifugation. This subcellular fr...

Discussion

The protocol presented here has been successfully used and continuously optimized for a long-time to determine the protein localization in the submitochondrial compartments^{13,14,18,21,22,23}. The reliability and reproducibility of this protocol are strongly dependent on the purity and integrity of mitochondrial preparations

Materials

Name	Company	Catalog Number	Comments
Bacto Peptone	BD	211677
Bacto Yeast extract	BD	212750
Beckman Ultra-Clear Centrifuge Tubes, 14 x 89 mm	Beckman Coulter	344059
Bovine serum albumin (BSA fatty acid free)	Sigma-Aldrich	A7030	Component of Homogenization buffer
DL-Dithiothreitol	Sigma-Aldrich	43815	Component of DDT buffer
D-Sorbitol	Sigma-Aldrich	S1876
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E9884
Galactose	Sigma-Aldrich	G0625
Glucose	Sigma-Aldrich	G7021
MOPS	Sigma-Aldrich	M1254
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	P7626	Used to inactivate proteinase K
Potassium phosphate dibasic	Sigma-Aldrich	P3786
Potassium phosphate monobasic	Sigma-Aldrich	P0662
Proteinase K	Sigma-Aldrich
Sucrose	Sigma-Aldrich	S8501
Trichloroacetic acid (TCA)	Sigma-Aldrich	T6399
Trizma Base	Sigma-Aldrich	T1503
Zymolyase-20T from Arthrobacter luteus	MP Biomedicals, Irvine, CA	320921	Used to lyse living yeast cell walls to produce spheroplast