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 more important, pore one.

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 high 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.

To begin, take 10 milligrams of freshly isolated crude yeast mitochondria and resuspend 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-cooled 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. 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 form the pellet, while the vesicles stay in the supernatant. Next, transfer the supernatant to fresh ultracentrifugation 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 two molar.

To adjust the sucrose concentration to 0.6 molars, add the appropriate MOPS, EDTA, PMSF, 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 immuno blotting. 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 in fractions 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 in fractions of intermediate density.