Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.
Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.
Mitochondria are organelles that exist in complex morphologies in different cell types and are recognized to possess an increasing array of functions that are critical for cellular health. As such, they can no longer be whittled down merely to energy-producing organelles. Mitochondria are key metabolic regulators, determinants of cell fate, and signaling hubs, the functions of which can serve as useful indicators of overall cellular health. In skeletal muscle cells, electron microscopy studies reveal the presence of geographically distinct subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, which exhibit a degree of connectivity1,2,3,4 that is now recognized to be highly dynamic and adaptable to changes in skeletal muscle activity levels, as well as with age and disease. Mitochondrial content and function in muscle can be assessed in numerous ways5,6, and traditional methods of organelle isolation have been applied to better understand the respiratory and enzymatic capacities (Vmax) of mitochondria distinct from the influence of the cellular milieu7,8. In particular, these traditional methods have revealed subtle biochemical distinctions between mitochondria isolated from subsarcolemmal and intermyofibrillar regions, belying possible functional implications for metabolism in these subcellular regions8,9,10,11.
The biogenesis of mitochondria is unique in requiring the contribution of gene products from both nuclear and mitochondrial DNA. However, the vast majority of these are derived from the nucleus since mtDNA transcription only leads to the synthesis of 13 proteins. Since mitochondria normally comprise >1000 proteins involved in diverse metabolic pathways, biogenesis of the organelle requires a tightly regulated means of import and assembly of precursor proteins from the cytosol into the various mitochondrial sub-compartments to maintain proper stoichiometry and function12,13. Nuclear-encoded proteins destined for mitochondria normally carry a mitochondrial targeting sequence (MTS) that targets them to the organelle and facilitates their sub-compartmental localization. Most matrix-bound proteins contain a cleavable N-terminal MTS, while those destined for the outer or inner mitochondrial membrane usually have internal targeting domains14. The import process is carried out by a set of diverse channels that provide multiple avenues for entry into the organelle13. The translocase of the outer membrane (TOM) complex shuttles precursors from the cytosol into the intermembrane space, where they are recognized by the translocase of the inner membrane (TIM) complex. This complex is responsible for importing nuclear-encoded precursors into the matrix, where proteases cleave the N-terminal targeting presequence. Proteins destined for the outer membrane can be directly inserted into this membrane through the TOM complex, while those destined for the inner membrane are inserted by a TIM protein, specifically TIM22. Following their import, proteins are further processed by resident proteases and chaperones and often combine to form larger complexes, such as those found in the electron transport chain.
Mitochondrial protein import itself also serves as a measurement of mitochondrial health, as this process relies on the presence of membrane potential and a source of energy in the form of ATP15. For example, when the membrane potential is dissipated, protein kinase PINK1 cannot be taken up by the organelle, and this leads to phosphorylation signals that trigger the onset of the degradation of the organelle through a pathway called mitophagy16,17. Under similar circumstances, when the import is impeded, the protein ATF5 cannot enter the organelle, and it subsequently translocates to the nucleus, where it serves as a transcription factor for the up-regulation of UPR gene expression18,19. Thus, measuring protein import efficiency can provide comprehensive insight into the health of the organelle, while the gene expression response can be used to indicate the degree of retrograde signaling to the nucleus.
Despite its obvious importance for the biogenesis of mitochondria and for cellular health in general, the import pathway in mammalian mitochondria is remarkably understudied. In this report, we describe the specific steps involved in measuring the import of precursor proteins into skeletal muscle mitochondria and provide data to illustrate the adaptive response of the import system to changes in muscle and disuse, illustrating the contribution of the protein import to the adaptive plasticity of skeletal muscle.
All animals used in these experiments are maintained in the animal care facility at York University. The experiments are conducted in accordance with the Canadian Council on Animal Care guidelines with approval from the York University Animal Care Committee (Permit: 2017-08).
1. Functional isolation of subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle
2. Mitochondrial protein import
We have extensively illustrated that this protocol is a valid assay for determining the rate of import into functional and intact isolated skeletal muscle mitochondria. In comparison to untreated conditions, the import of typical precursor proteins such as malate dehydrogenase (MDH) into the matrix is sensitive to membrane potential because it can be inhibited by valinomycin, a respiratory chain uncoupler (Figure 2A). Import is also impeded when mitochondrial inner and outer membranes are solubilized in the presence of the detergent Triton X-100. The import process is sensitive to the presence of external ATP, which serves to unfold precursor proteins for translocation across the membranes, and is tightly controlled by the rate of respiration and ATP provision (data not shown20). Distinct differences in the import are also observed between intermyofibrillar and subsarcolemmal mitochondrial fractions, in part due to variations in protein import machinery expression, as well as the respiratory rate between these mitochondria (Figure 2B).
Mitochondria in skeletal muscle are highly dynamic organelles that respond readily to changes in energy demand. Mitochondrial content in the muscle increases following periods of chronic exercise or in response to electrical stimulation-induced contractile activity (see for review21). For example, 7 days of chronic contractile activity of rat skeletal muscle enhances import into the OM and matrix by 1.6-fold and 1.4-fold, respectively22 (Figure 3A, 3B). These changes in mitochondrial content are brought about, in part, by alterations in the capacity of the mitochondrial protein import system. Indeed, a close relationship between the import rate of precursor proteins and a good estimate of mitochondrial content, as measured by the complex IV marker cytochrome oxidase under control or denervated conditions, can be illustrated23 (Figure 3C).
The adaptability of the import system in muscle to alterations in contractile activity suggests that exercise could be used as a treatment to resolve defects in the import pathway if identified. During the investigation of mitochondrially-mediated apoptosis using Bax-Bak double knockout animals, we noticed that the reduced mitochondrial content in the muscles of these experimental animals was accompanied by a decrease in the import of precursor proteins into the matrix. We then investigated the possibility that exercise could restore this import capacity. Indeed, following 6 weeks of voluntary wheel run training, protein import was restored to control levels in the knockout animals24, illustrating the adaptive plasticity of the import pathway to rescue mitochondrial content and function (Figure 4).
Figure 1: Schematic of the workflow for isolating SS and IMF mitochondria from skeletal muscle based on the previous work8. This protocol allows the isolation of functional mitochondria based on their geographic location within skeletal muscle. SS mitochondria are more rapidly and easily liberated, while the IMF mitochondria require a further digestion step with protease to untangle them from the myofibrils. Note that the isolation of these subfractions can be done in tandem. An updated, similar procedure has recently been published25. Please click here to view a larger version of this figure.
Figure 2: Protein import into the mitochondrial matrix. (A) Normal rates of import for MDH are shown in SS and IMF mitochondria (lanes 4 and 7) and the translation product of precursor MDH (lane 1). The lower band represents the imported mature MDH. Addition of valinomycin inhibits MDH protein import into the matrix of SS and IMF mitochondria, as this uncoupler dissipates the membrane potential (lanes 2 and 5). Triton-X is a detergent that solubilizes the inner membrane, thereby inhibiting MDH import into these subfractions (lanes 3 and 6). Protein import was carried out for increasing time durations, 4 min, 7 min, 10 min, 15 min, and 30 min. These data illustrate that import is a time-dependent process and also that SS and IMF mitochondria have different rates or capacities for import (B). TL, translation lane; VAL, valinomycin; TRI, Triton-X; CTL, control; SS, subsarcolemmal; IMF, intermyofibrillar. This figure was modified from Takahashi M & Hood DA20. Please click here to view a larger version of this figure.
Figure 3: Protein import is adaptable and tightly linked to estimates of mitochondrial content. Sprague Dawley rats were subjected to electrical stimulation to induce contractile activity, a model of exercise training. (A) Tom40 import into the OM was 1.6-fold higher in muscle from chronically stimulated animals compared to controls at any given time point. (B) OCT import into the mitochondrial matrix was increased at every time point of incubation, and overall, this resulted in a 1.4-fold increase in mitochondria from chronically stimulated muscle. (C) Import is positively correlated with an index of mitochondrial content, as assessed by COX activity, r = 0.69. These measurements were taken from animals that were subjected to denervation, which has been shown to decrease mitochondrial content and the rate of import. Con, control; Den, denervated; Stim, stimulated; TL, translation lane; * p < 0.05. Figures 3A and 3B were adapted from Joseph A-M & Hood DA22 and 3C from Singh B & Hood DA23. Please click here to view a larger version of this figure.
Figure 4: Training rescues import defects in vivo. Bax/Bak double knockout animals exhibit reduced protein import into the mitochondrial matrix by 37%. Six weeks of voluntary wheel running rescued the import defect to control levels. WT, wildtype; DKO, double knockout; TL, translation lane; * p < 0.05 main effect of genotype; ¶ p < 0.05 main effect of training. This figure was adapted from Zhang Y et al.24. Please click here to view a larger version of this figure.
Buffer 1: | Buffer 1 + ATP: | Buffer 2: | Resuspension medium: | Nagarse protease |
100 mM KCl | 100 mM KCl | 100 mM KCl | 100 mM KCl | 10 mg/mL in Buffer 2 |
5 mM MgSO4 | 5 mM MgSO4 | 5 mM MgSO4 | 10 mM MOPS | NOTE: Make fresh and keep on ice |
5 mM EDTA | 5 mM EDTA | 5 mM EGTA | 0.2% BSA | |
50 mM Tris | 50 mM Tris | 50 mM Tris | ||
1 mM ATP | 1mM ATP |
Table 1: Buffers and resuspension media.
% of the reaction volume | 1 Reaction mix | |
Reticulocyte Lysate | 64.10% | 11.8 µL |
Amino acids (-methionine) | 2.20% | 0.4 µL |
Sterile H2O | 21.60% | 3.97 µL |
35S-methionine | 7.20% | 1.33 µL |
mRNA | 5.40% | 1.0 µL |
Total Volume | 18.5 µL | |
NOTE: | ||
1) Add 35S-methionine last; | ||
2) Thaw the lysate slowly on ice, and limit freeze/thaw cycles to two. It is recommended that the lysate be aliquoted upon arrival | ||
3) The volume of mRNA can be adjusted to optimize translational efficiency by altering the sterile H2O volume accordingly. |
Table 2: Translation reaction mix
Mitochondria are uniquely dependent on the expression and coordination of both the nuclear and mitochondrial genomes for their synthesis and expansion within cells. However, the nuclear genome encodes the vast majority (99%) of the mitochondrial proteome, and this underscores the importance of the protein import machinery in supporting mitochondrial biogenesis. Import also serves as an important signaling event, as failure to import can promote the initiation of the unfolded protein response and/or mitophagy15,16,26. Since import relies on the expression of multiple functional channels and chaperones, an intact membrane potential, as well as the availability of ATP, the evaluation of mitochondrial protein import can provide valuable insight into the health and energy status of the organelle.
The evaluation of protein import capacity into mitochondria requires the isolation of the organelle from the surrounding tissue using standard differential centrifugation techniques. Import is evaluated in the same manner as one would evaluate the Vmax of mitochondrial enzyme activities, in a highly controlled manner dependent on time and substrate concentration and independent of cellular cytosolic influences. The isolation method presented here, or similar renditions of it, have been used for many years to evaluate mitochondrial respiration and enzyme activities8,9,10,11. This technique is not without its limitations, as it disrupts the normal morphology of mitochondria as they exist in situ27, which in skeletal muscle is highly complex, ranging from small spheroid structures, to a highly branched reticular network3, depending on their subsarcolemmal or intermyofibrillar location within the muscle cell. In addition, the use of the protease nagarse has come under scrutiny3,28. An update to this method, using trypsin instead of nagarse, has recently been published25, and trypsin has been used by others to isolate muscle mitochondria29. Indeed, any alternative isolation method that yields functionally intact mitochondria can be used, including techniques that do not employ proteases30 or those designed for small biopsy-sized samples from human muscle31. The method presented here has the advantage of isolating SS mitochondria quickly and easily, but a greater yield and purity of mitochondria can be obtained through the isolation of the IMF subfraction. If done carefully, this isolation protocol can result in functionally intact organelles with a high respiratory control ratio, indicative of appropriate rates of state 3 and 4 respiration8.
It is also imperative for the measurement of mitochondrial protein import that these organelles maintain their membrane potential, as import across the inner membrane is dependent on this electrophoretic proton motive force, which serves to attract positively charged precursor sequences and help mediate the transport of the precursor into the negatively charged matrix space. Under such conditions, comparisons of mitochondrial function and import can be evaluated between physiologically relevant experimental situations, such as exercise, aging, and muscle disuse, for example. In this respect, previous work has shown that import is a highly adaptable pathway that responds to altered states of muscle use and disuse and is sensitive to inhibition via excessive ROS emission10,22,23,32. This plasticity is due, in part, to adaptive changes in the expression of the protein import machinery components. Since this technique relies on the isolation of mitochondria, any regulation on the import pathway that may stem from cytosolic factors or inter-organelle crosstalk is removed from the interpretation of the experiment. This is both a limitation and a strength of the technique, as conclusions can be made of the capacity of the import pathway itself (similar to the Vmax of enzyme activity), but transient or external signals that may occur with the experimental model may be lost. To circumvent this, it is possible to incubate the import reaction with a sample of the cytosolic fraction, isolated as described above, in order to evaluate changes in the cytosolic environment that may influence import rates, as done previously22. In addition, import machinery components are subject to acute, post-translational modifications that can alter their functions. Recent work has shown that phosphorylation of specific TOM import receptors can be linked to mitophagy33. Indeed, an area of research that warrants more attention is the acute modulation of the import process via intrinsic signaling pathways mediated by ROS or by covalent modification of import receptors and chaperones, as documented in yeast and other lower organisms34,35.
Protein import into mitochondria represents a gateway to adaptive organelle growth and is a sensitive indicator of mitochondrial health. Understanding how this process is regulated can shed light on the regulation of mitochondrial biogenesis, UPR signaling, and the initiation of mitophagy. Mitochondrial protein import is not a widely studied process in mammalian experimental models, and the development of the vast potential of research in this area could help us achieve a greater understanding of diseases in which mitochondrial dysfunction is apparent or represent an attractive therapeutic target to promote mitochondrial health.
The authors have nothing to disclose.
The authors would like to thank Dr. G.C. Shore of McGill University, Dr. A. Strauss of the Washington School of Medicine, and Dr. M.T. Ryan of La Trobe University for the original donations of expression plasmids that were used for this research. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to D. A. Hood. D. A. Hood is also the holder of a Canada Research Chair in Cell Physiology.
0.2% BSA | Sigma | A2153 | |
35S-methionine | Perkin Elmer | NEG709A500UC | Purchase requires a valid radioisotope permit |
ATP | Sigma | A7699 | |
Blotting paper; Whatman 3MM CHR Paper | Thermo Fisher | 05-714-5 | |
Cassette for film | Kodak | Kodak Xomatic | |
Centrifugation Tube | Thermo Fisher | 3138-0050 | |
Chloroform | Thermo Fisher | C298-4 | |
DTT | Sigma | D9779-5G | |
EDTA | BioShop | EDT002 | |
EGTA | Sigma | E4378 | |
Gel Dryer | BioRad | Model 583 | |
Gel Drying Kit | Sigma or BioRad | Z377570-1PAK or OW-GDF-10 | Various options are commercially available through many companies, these are just as few examples. |
Glycerol | Caledon Laboratory Chemicals | 5350-1-40 | |
HEPES | Sigma | H3375 | |
High Speed Centrifuge | Beckman Coulter | Avanti J-25 Centrifuge | |
Homogenizer | IKA | T25 Digital Ultra Turrex | |
Isoamylalcohol, or 3-methylbutanol | Sigma | I9392 | |
KAc | BioShop | POA301.500 | |
KCl | Sigma | P3911 | |
M7G | New England Biolab | S1404S | Dilute with 1000ul 20mM HEPES to make 1mM stock |
MgCl | BioShop | MAG510 | |
MgSO4 | Thermo Fisher | M65-500 | |
MOPS | BioShop | MOP001 | |
NaCl | BioShop | SOD001 | |
NTP | Thermo Fisher | R0191 | |
OCT Plasmid | – | – | Donated from Dr. G. C. Shore, McGill University, Montreal, Canada; alternative available through Addgene, plasmid #71877 |
pGEM4Z/hTom40 Plasmid | – | – | Donated from Dr. M. T. Ryan, La Trobe University, Melbourne, Australia |
pGMDH Plasmid | – | – | Donated from Dr. A. Strauss, Washington University School of Medicine |
Phenol | Sigma | P4557 | |
Phenol:Chloroform:Isoamyalcohol | Sigma | P3803 | Can also be made with the ratio provided |
Phosphorus Film | Fujifilm | BAS-IP MS 2025 | |
Rabbit reticulocyte lysate | Promega | L4960 | Avoid freeze-thaw; aliquot lysate upon arrival; amino acids are provided in the kit as well |
RNAsin | Promega | N2311 | |
Rotor for High Speed Centrifuge | Beckman Coulter | JA-25.50 | |
SDS | BioShop | SDS001.500 | Caution: harmful if ingested or inhaled, wear a mask. |
Sodium acetate | Bioshop | SAA 304 | |
Sodium Carbonate | VWR | BDH9284 | |
Sodium salicylate | Millipore Sigma | 106601 | |
Sorbitol | Sigma | S6021 | |
SP6 RNA Polymerase | Promega | P1085 | |
Spectrophotometer | Thermo Fisher | Nanodrop 2000 | |
Spermidine | Sigma | S-2626 | |
Sucrose | BioShop | SUC507 | |
T7 RNA Polymerase | Promega | P2075 | |
Tabletop Centrifuge | Thermo Fisher | AccuSpin Micro 17 | |
Trichloroacetic acid | Thermo Fisher | A322-500 | |
Tris | BioShop | TRS001 | |
β-mercaptoethanol | Sigma | M6250-100ML |