generating submitochondrial vesicles from saccharomyces cerevisiae mitochondria to isolate mitochondrial contact sites

Isolation of Mitochondrial Contact Sites

Mitochondria perform a variety of different functions in eukaryotes, with the most well-known being the production of ATP through oxidative phosphorylation. Other functions include the production of iron-sulfur clusters, lipid synthesis, and in higher eukaryotes, Ca2+ signaling, and the induction of apoptosis1,2,3,4. These functions are inseparably linked to their complex ultrastructure.
The mitochondrial ultrastructure was first described by electron microscopy5. It was shown that mitochondria are rather complex organelles consisting of two membranes: the mitochondrial outer membrane and the mitochondrial inner membrane. Thus, two aqueous compartments are formed by these membranes: the intermembrane space and the matrix. The mitochondrial inner membrane can be even further divided into different sections. The inner boundary membrane stays in close proximity to the outer membrane, and the cristae form invaginations. So-called crista junctions connect the inner boundary membrane and the cristae (Figure 1). Furthermore, electron micrographs of osmotically shrunken mitochondria reveal that sites exist at which the mitochondrial membranes are tightly connected6,7. These so-called contact sites are formed by protein complexes spanning the two membranes (Figure 1). It is thought that these interaction sites are essential for cell viability due to their importance for the regulation of mitochondrial dynamics and inheritance, as well as the transfer of metabolites and signals between the cytosol and the matrix8.
The MICOS complex in the mitochondrial inner membrane is probably the best characterized and the most versatile contact site-forming complex. MICOS was described in yeast in 2011, and it consists of six subunits9,10,11: Mic60, Mic27, Mic26, Mic19, Mic12, and Mic10. These form a complex of approximately 1.5 MDa that localizes to the crista junctions9,10,11. The deletion of either core subunit, Mic10 or Mic60, leads to the absence of this complex9,11, meaning these two subunits are essential for the stability of MICOS. Interestingly, MICOS forms not only one but multiple contact sites with various mitochondrial outer membrane proteins and complexes: the TOM complex11,12, the TOB/SAM complex9,12,13,14,15,16, the Fzo1-Ugo1 complex9, Por110, OM4510, and Miro17. This strongly indicates that the MICOS complex is involved in various mitochondrial processes, such as protein import, phospholipid metabolism, and the generation of the mitochondrial ultrastructure18. The latter function is probably the major function of&#160;MICOS, as the absence of the MICOS complex induced through the deletion of MIC10&#160;or MIC60&#160;leads to an abnormal mitochondrial ultrastructure that virtually completely lacks regular cristae. Instead, internal membrane vesicles without connection to the inner boundary membrane accumulate19, 20. Importantly, MICOS is conserved in form and function from yeast to&#160;human21. The association of mutations in MICOS subunits with severe human diseases also emphasizes its importance for higher eukaryotes22,23. Although MICOS is highly versatile, additional contact sites must exist (based on our unpublished observations). Indeed, several other contact sites have been identified, for instance, the mitochondrial fusion machineries Mgm1-Ugo1/Fzo124,25,26&#160;or Mdm31-Por1, which is involved in the biosynthesis of the mitochondrial-specific phospholipid cardiolipin27. Recently, we improved the method that led us to the identification of MICOS to identify Cqd1 as part of a novel contact site formed with the outer membrane complex Por1-Om1428. Interestingly, this contact site also seems to be involved in multiple processes such as mitochondrial membrane homeostasis, phospholipid metabolism, and the distribution of coenzyme Q28,29.
Here, we used a variation of the previously described fractionation of mitochondria9,30,31,32,33. Osmotic treatment of mitochondria leads to the disruption of the mitochondrial outer membrane and to a shrinkage of the matrix space, leaving the two membranes only in close proximity at contact sites. This allows for the generation of vesicles that consist exclusively of mitochondrial outer membrane or mitochondrial inner membrane or at contain contact sites of both membranes through mild sonication. Due to the mitochondrial inner membrane possessing a much higher protein-to-lipid ratio, mitochondrial inner membrane vesicles exhibit a higher density compared to mitochondrial outer membrane vesicles. The difference in density can be used to separate the membrane vesicles through sucrose buoyant density gradient centrifugation. Thus, the mitochondrial outer membrane vesicles accumulate at low sucrose concentrations, while the mitochondrial inner membrane vesicles are enriched at high sucrose concentrations. The vesicles containing contact sites concentrate at intermediate sucrose concentrations (Figure 2). The following protocol describes this improved method, which requires less specialized equipment, time, and energy compared to our previously established one32, in detail and provides a useful tool for the identification of possible contact site proteins.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

An Improved Method to Isolate Mitochondrial Contact Sites

We aim to understand the molecular function of mitochondrial contact sites, allowing the communication between the two mitochondrial membranes. This actually is a prerequisite to grasp the function for mitochondrial biogenesis and thus, cell viability. We discovered a new contact site between the mitochondrial inner and outer membrane created by Cqd1 and Om14-Por1.Our data suggests that this site might assist in lipid import like phosphatidic acid. Based on the identification of the MICOS complex, we contributed to get a deeper understanding of how mitochondrial membrane architecture is generated. This allowed us to develop a working model that explains how the two forms of mitochondrial cristae, lamellar and tubular cristae are formed.Our research was done in yeast in the past. However, we are convinced that the formation of correct mitochondrial architecture is important for higher eukaryotes too. So the next step in our research is analyzing the role of mitochondrial architecture elements in more complex models such as primary neurons.

Watch this Scientific Journal Video about Generating Submitochondrial Vesicles from  Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites at JoVE.com

Generating Submitochondrial Vesicles from  Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites  

Generating Submitochondrial Vesicles from Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites

To begin, take 10 milligrams of freshly-isolated crude yeast mitochondria and re-suspend them in 1.6 milliliters of SM buffer at four degrees celsius by pipetting. Then, transfer the mitochondrial suspension to a pre-cooled 100-milliliter Erlenmeyer flask. Slowly, add 16 milliliters of the swelling buffer, using a 20-milliliter glass pipette while applying constant mild stirring on ice.Incubate the samples under constant mild stirring for 30 minutes on ice. Then, slowly add five milliliters of 2.5-molar sucrose solution using a five-milliliter glass pipette, allowing the inner membrane to shrink. Incubate the samples under mild stirring for 15 minutes on ice.To generate sub-mitochondrial membrane vesicles, transfer the mitochondrial suspension to a pre-cool rosette cell, and sonicate the suspension at 10%amplitude for 30 seconds while cooling the rosette cell in an ice bath. Rest the suspension for 30 seconds in an ice bath.

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Biochemistry

91 vues.  Ludwig-Maximilians University Munich. Pour commencer, prenez 10 milligrammes de mitochondries de levure brutes fraîchement isolées et mettez-les en suspension dans 1,6 millilitre de tampon SM à quatre degrés Celsius par pipetage. Ensuite, transférez la suspension mitochondriale dans un erlenmeyer pré-refroidi de 100 millilitres. Lentement, ajoutez 16 millilitres de tampon gonflant, à l’aide d’une pipette en verre de 20 millilitres tout en remuant doucement et constamment sur de la glace.Incuber les échantillons...

Vidéo: Generating Submitochondrial Vesicles from  Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites  

an improved method to isolate mitochondrial contact sites

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of...

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separation and analysis of submitochondrial vesicles to identify mitochondrial contact site proteins

Separation and Analysis of Submitochondrial Vesicles to Identify Mitochondrial Contact Site Proteins

assessment of submitochondrial protein localization in budding yeast saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

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...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

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This video describes the procedure for isolating mitochondria from human ovarian cancer tissues using differential-speed centrifugation in combination with density gradient centrifugation. The isolated mitochondria can be used for proteomic analysis of human ovarian cancer mitochondrial...

Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample

Then, use clean ophthalmic scissors to fully mince the tissue into pieces that are approximately one cubic millimeter and transfer the minced tissues into a 50-milliliter centrifuge tube. Add 13.5 milliliters of mitochondrial isolation buffer containing nagarse at a concentration of 0.2 milligrams per milliliter. Use an electric homogenizer to homogenize the minced tissues for two minutes at 4 degrees...

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Labelling and Visualization of Mitochondrial Genome Expression Products in Baker&#039;s Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate...

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visualization and quantification of endogenous intra-organelle protein interactions at er-mitochondria contact sites by proximity ligation assays

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at...

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Locus-Specific Chromatin Purification from Saccharomyces cerevisiae  

Locus-Specific Chromatin Purification from Saccharomyces cerevisiae

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

coupled assays for monitoring protein refolding in saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae.  Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality...

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microarray analysis for saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing...

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Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

dissection of saccharomyces cerevisiae asci

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of...

Dissection of Saccharomyces Cerevisiae Asci

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media...

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cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

isolation of mitochondria for mitochondrial supercomplex analysis from small tissue and cell culture samples 

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples 

single-copy gene locus chromatin purification in saccharomyces cerevisiae

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces...

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genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

To begin, take the prepared yeast sub-mitochondrial vesicles and centrifuge the generated vesicles and remaining intact mitochondria at 20, 000 G for 20 minutes at four degrees Celsius. The intact mitochondria formed the pellet while the vesicles stay in the supernatant. Next, transfer the supernatant to fresh ultra centrifugation tubes.Load a cushion of 0.3 milliliters of 2.5 molar sucrose solution at the bottom of the tube using a one milliliter syringe equipped with a cannula, and centrifuge at 118, 000 G for 100 minutes at four degrees Celsius. After centrifugation, the vesicles will appear as a disc on the top of the sucrose cushion. Discard approximately 90%of the supernatant.To harvest the concentrated vesicles, resuspend them in the remaining buffer, including the 2.5 molar sucrose by pipetting. Transfer the suspension to an ice cold Dounce homogenizer and homogenize the suspension using a polytetrafluoroethylene potter with at least 10 strokes. Next, prepare an 11 milliliter step gradient with each layer containing 2.2 milliliters of sucrose solution.Calculate the sucrose concentration using this formula. Add the highest sucrose concentration to the centrifugation tube and place it at minus 20 degrees Celsius until the layer is completely frozen. Measure the sucrose concentration of the samples using a refractometer.If a refractometer is unavailable, assume the sucrose concentration is 2 molar. To adjust the sucrose concentration to 0.6 molars, add the appropriate MOPS, E-D-T-A, P-M-S-F, and protease inhibitor cocktail at pH 7.4. Carefully load the samples on the sucrose gradient to avoid the disturbance of the gradient.Centrifuge the vesicles at 200, 000 G and four degree Celsius for 12 hours. If possible, set slow acceleration and deceleration to avoid disruption of the gradient. Then harvest the gradient from top to bottom in 700 microliters fractions, using a one milliliter pipette.Resulting in 17 fractions, which provides a sufficient resolution. Next, perform two sequentially executed trichloroacetic acid precipitations by adding 200 microliters of 72%trichloroacetic acid to the individual fractions and mixing until the solution is homogeneous. Incubate the fractions for 30 minutes on ice and pellet the precipitated proteins by centrifuging at 20, 000 G and four degrees Celsius for 20 minutes.Discard the supernatant. Add 500 microliters of 28%trichloroacetic acid solution. Mix well and repeat the centrifugation step.Finally, analyze the fractions by SDS-PAGE and immunoblotting. The importance of mild sonication is presented here. The mitochondrial outer membrane marker Tom40 was enriched in the early low density fractions and was virtually absent in the later ones.The mitochondrial inner membrane marker Tim17 was concentrated in high density fractions. In contrast, the contact site protein Mic60 accumulated infractions of intermediate sucrose concentration, indicating the successful generation and subsequent separation of the various membrane vesicles. In contrast, with harsher sonication conditions, the mitochondrial outer membrane marker Tom40 could be detected in lower fractions of the gradient.Additionally, there was a significant amount of Tim17 infractions of intermediate density.