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. Author manuscript; available in PMC: 2017 Aug 8.
Published in final edited form as: Methods Mol Biol. 2016;1376:133–140. doi: 10.1007/978-1-4939-3170-5_11

Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor

Abasha Lewis, Shang-Yi Tsai, Tsung-Ping Su
PMCID: PMC5547822  NIHMSID: NIHMS880685  PMID: 26552680

Abstract

The interface between the endoplasmic reticulum (ER) and mitochondria referred to as the MAM (mitochondria-associated ER membrane) plays important roles in many physiological functions. A specific marker for this important entity of cellular structure is urgently needed. Thus, we propose in this method chapter that the membrane-bound ER chaperone sigma-1 receptor serves as an ideal marker for the MAM. We describe in detail the preparation and purification of the MAM by using the sigma-1 receptor as the marker and demonstrate the uniqueness of this marker by using a variety of cells, peripheral and neuronal.

Keywords: Sigma-1 receptor, Chaperone, Endoplasmic reticulum, Mitochondria, MAM, SDS/PAGE electrophoresis, Fractionation

1 Introduction

Since its detection in the 1970s, the specialized area of the ER in physical contact with the outer mitochondrial membrane, termed mitochondria-associated ER membrane (MAM) [1–7], has emerged as a critical signaling junction within the cell. In fact, the transient contact the MAM provides [8] is used to facilitate critical cellular processes including phospholipid exchange, Ca2+ signaling, autophagosome formation, and cellular morphology [9], and has been implicated in several disease models. Just recently, point mutations in the presynaptic protein α-synuclein associated with Parkinson disease was determined to have reduced association with the MAM, in comparison to wild-type α-synuclein, and coincident with a decrease in MAM function and increased fragmentation of mitochondria [10]. Abrogation of MAM functioning has also been implicated in Alzheimer’s disease [11–13], diabetes [14–16], and cancer [17].

Due to the increasingly critical implications of MAM localized proteins in the study of neurodegenerative disease, we provide a subcellular fractionation assay for the isolation of MAM by taking advantage of differential centrifugation. However, validation of isolated MAM can be problematic. Previous studies have identified several ER-resident proteins enriched at the MAM [9]; however, most of these proteins are also highly detectable in the bulk ER and other cellular compartments. Although phosphatidylethanolamine-N-methyltransferase-2 has emerged as a specific marker for the MAM, it is only reliable in liver and primary hepatocytes [18]. Thus, after careful examination of several MAM-enriched proteins, we show that sigma-1 receptor (Sig-1R) is the most reliable protein marker for this subcellular local in various mammalian cell and tissue types.

2 Materials

2.1 Cell and Tissue Preparation

  1. Cell scraper.

  2. Phosphate buffered saline (PBS): 13.7 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. Adjust pH to 7.4 and autoclave.

  3. Surgeon’s scalpel.

  4. Hank’s balanced salt solution (HBSS) without Ca2+ and Mg2+: 13.7 mM NaCl, 5.33 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4. Adjust pH to 7.4 and autoclave.

2.2 MAM Isolation Assay

  1. Teflon glass homogenizer.

  2. Pasteur pipettes, fire-polished.

  3. Ultracentrifuge with swinging bucket and fixed angle rotors.

  4. Large (16 × 76 mm) and small (13 × 51 mm) polycarbonate thick-walled tubes are preferred for use with the rotors.

  5. Homogenization buffer (H-B): 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, and 0.25 M sucrose. Store at 4 °C.

  6. Isolation medium (I-M): 5 mM HEPES, pH 7.4, 250 mM mannitol, 0.5 mM EGTA. Store at 4 °C.

  7. Isolation medium 2 (I-M2): 25 mM HEPES, pH 7.4, 225 mM mannitol, 1 mM EGTA. Store at 4 °C.

  8. Percoll medium: 25 mM HEPES, pH 7.4, 225 mM mannitol, 1 mM EGTA, 30 % Percoll (v/v). Make fresh before experiment.

  9. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and western blotting equipment.

  10. Optional: 10,000 MWCO Centrifugation Filtration Unit.

3 Methods

3.1 Cell and Tissue Preparation

3.1.1 Cell Preparation

  1. Culture adherent cells in 15-cm dishes until 90–100 % confluent (see Note 1).

  2. Place cells on ice and wash once with ice-cold PBS.

  3. Collect cells in ice-cold PBS by scraping and place into conical tube.

  4. Centrifuge cells at 500 × g to pellet and discard supernatant. Remove excess supernatant by inverting the tube on a Kimwipes for 30–60 s.

3.1.2 Tissue Preparation

  1. Use standard procedures to dissect tissue and place on ice(see Note 2).

  2. Using a surgeon’s scalpel, cut the tissue into small pieces and place into conical tube with HBSS.

  3. Centrifuge tissue at 500 × g to pellet and discard supernatant. Remove excess supernatant by inverting the tube on a Kimwipes for 15–30 s.

3.2 MAM Isolation Assay

To avoid protein degradation during the process, the subcellular fractions should be prepared on ice and centrifuged at 4 °C. Store fractions at −80 °C, if not used immediately.

  1. Suspend pelleted cells/tissue in H-B (approximately 9 v/w of wet pellet) and homogenate very slowly with a Teflon glass homogenizer for 20–30 strokes (see Note 3). Return homogenate to original conical tube.

  2. Centrifuge homogenate at 600 × g for 5 min. Remove supernatant and place into new conical or eppendorf tube(s). Suspend pellet in H-B and homogenate again (7–15 strokes).

  3. Centrifuge homogenate at 600 × g for 5 min. Combine resulting supernatant with previous one from step 2. Suspend pellet containing cell debris and nuclei in 0.5 ml I-M and store as P1 fraction.

  4. Centrifuge supernatant at 10,300 × g for 20 min. Collect supernatant containing microsome and cytosol in large polycarbonate thick-walled tube(s) and place on ice until step 9. Thoroughly suspend pellet containing crude mitochondria (mitochondria with intact MAM) in 500 μl of I-M.

  5. Layer the crude mitochondria suspension on top of 3 ml of Percoll medium (Fig. 1) in a small polycarbonated thick-walled tube (see Note 4). Centrifuge at 95,000 × g for 30 min using a swinging bucket rotor with deceleration set to zero as not to disturb the gradient.

  6. Collect the MAM and mitochondria (Fig. 2) using fire-polished Pasteur pipettes (see Note 5). The MAM is observed as a diffuse white layer above the mitochondria. Dense bands containing mitochondria are recovered at approximately ¾ – ⅔ of the way down the tube.

  7. Dilute the MAM with five times volume I-M2 and centrifuge at 6300 × g for 10 min. Place supernatant containing the MAM into a large polycarbonate thick-walled tube. Optional: Resuspend pellet in I-M2 and store as Crude MAM (MAM attached to mitochondria or aggregated MAM membranes), otherwise, discard.

  8. Centrifuge the MAM and microsome/cytosol from step 4 at 100,000 × g for 1 h using a fixed angle rotor.

  9. Dilute mitochondria with five times I-M. Wash three times by centrifugation at 10,500 × g for 10 min (see Note 6). Discard all of the supernatant. Collect pelleted Mitochondria fraction by suspending in I-M2 (50–200 μl to obtain 0.5–2 μg/μl protein concentration).

  10. Collect the MAM: The MAM appears as a loose floating white material (Fig. 3). Gently remove most of the supernatant. Using a fire-polished Pasteur pipette, carefully collect the MAM and suspend in H-B (50–200 μl to obtain 0.5–2 μg/μl protein concentration; see Note 7).

  11. Collect microsome and cytosolic fractions: Collect supernatant containing Cytosolic fraction (see Note 8). Add 200 μl of H-B to tube and suspend pelleted Microsome fraction by sonicating on ice for 10 s (repeat if necessary).

  12. Subcellular fractions (P1, Mito, MAM, P3, Cyto) are analyzed by SDS-PAGE and western blot analysis (Fig. 4).

Fig. 1.

Fig. 1

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Crude mitochondrial fraction layered over Percoll medium

Fig. 2.

Fig. 2

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Ultracentrifugation was used to separate mitochondria and MAM using a Percoll gradient. (a) The MAM can be found as a diffuse white band towards the top of the tube, while (b) mitochondria are found in several dense bands layered below. (c) Cellular debris collects on the bottom of the tube

Fig. 3.

Fig. 3

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Ultracentrifugation was used to pellet the purified MAM and is found as a loose white floating material at the bottom of the tube

Fig. 4.

Fig. 4

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Western blotting analysis of subcellular fractions collected by the MAM isolation assay protocol in (a) HEK cells. Known nuclear (NP62), mitochondrial (ATP synthase, Cytochrome c), MAM-enriched (Sig-1R, BiP, IP3 R3), and ER (ERp57) proteins were used to validate the fractions. Of the MAM-enriched proteins visualized, Sig-1R is the most reliable and is significantly less detectable in other cellular compartments. Cellular distributions of MAM-enriched proteins, Sig-1R and BiP, are also shown for (b) CHO cells, (c) Astrocytes, and (d) Neurons. Overall, Sig-1R was the most reliable protein marker for the MAM when compared to other MAM-enriched proteins

Acknowledgments

This work is supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health, of the Department of Health and Human Services of the United States of America.

Footnotes

1

Four to six 15-cm dishes are recommended to obtain an adequate protein concentration in each fraction; however, the researcher should optimize for the number of cells.

2

Standard dissection procedures can be found in [19].

3

Homogenization should be done very slowly and thoroughly as not to destroy the mitochondria and cause dissociation of the MAM. One stroke includes both the up and down movement.

4

Tube size may vary depending on the rotor used. The researcher should optimize the volume of Percoll medium so that it fills approximately ⅔ of the tube. It is also important that the two layers do not significantly mix. Gently layer crude mitochondria on top of Percoll medium so that the interface between the two layers is viewed as a sharp line.

5

Take care when collecting MAM and mitochondria. Place the tip of the fire-polished Pasteur pipette on the wall of the tube and collect from the top of the layer to avoid contamination.

6

This washing step is necessary to remove Percoll medium from the sample. In the presence of Percoll; however, the mitochondrial fraction will not pellet. Thus, after each centrifugation only remove the top ⅖ – ⅔ of the supernatant to ensure that mitochondria are retained. Washing is complete when a pellet is visible. All of the supernatant can be removed at this time.

7

Take particular care when collecting the MAM so that only the pellet is recovered. Further dilution of the MAM in H-B may not be necessary. It is up to the research to optimize for desired concentration.

8

Optional: The cytosolic fraction can be concentrated using a 10,000 MWCO centrifugal filtration unit.

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