This protocol is designed to determine the submitochondrial localization of yeast mitochondrial proteins, which is considered as a fundamental step during mitochondrial protein function elucidation. The protocol is suitable for our yeast strains maintained in different growth conditions. Representing a powerful tool for research as studying mitochondria.
To begin with, streak cells from glycerol stock stored at 80 C, onto a YPD agar plate to isolate single colonies from the strain of interest. And incubate the plate at 30 C for 2 to 3 days. Prepare a starter culture by inoculating 2 to 3 individual colonies from YPD agar plate in a 100 ml Erlenmeyer flask containing 10 to 30 ml of YPGal medium.
Incubate the flask at 30 C for 24 hours with vigorous shaking at 180 to 200 rpm. Dilute the starter culture into 1 L of fresh YPGal medium to an optical density less than 0.1 at 600 nm. Cultivate the cells at 30 C with vigorous shaking at 180 to 200 rpm for about 12 hours, until optical density reaches 1 to 1.5.
Perform the isolation of highly purified mitochondria as described in the text. And determine the protein concentration of the highly purified mitochondrial preparation, using the Bradford assay, following the manufacturer's instructions. Adjust the protein concentration to 10 mg/ml with ice-cold SEM buffer.
Transfer 40 l of highly purified mitochondria into four 1.5 ml pre-chilled and labeled microcentrifuge tubes. Add 360 l of SEM buffer in the first and second tubes. And 360 l of EM buffer in the third and fourth tubes.
Using the pipetting scheme, as per the text, add 4 L of freshly prepared proteinase K in the second and fourth tube. After mixing all the tubes gently, incubate on ice for 30 minutes with occasional mixing. To stop proteinase K activity add 4 L of 200 mM PMSF to all four tubes.
Centrifuge the tubes at 20, 000 x g for 30 minutes at 4 C.And collect the supernatant, without disturbing the pellet, into a new 1.5 ml pre-chilled labeled microcentrifuge tube. Next, resuspend the pellet in 400 l of ice-cold SEM buffer. To inactivate the possible traces of proteinase K, precipitate the collected supernatant and resuspend the pellet with trichloroacetic acid to a final concentration of 10%Incubate the tubes on ice for 10 minutes.
Centrifuge the trichloroacetic acid treated samples for 10 minutes at 12, 000 x g at 4 C.And after removing the supernatant, resuspend the pellet in 200 l of sample buffer. If the bromophenol blue turns yellow, add 1 to 5 l of 1 M Tris base, until it turns blue. Add 4 l of 200 mM PMSF to all the tubes.
Store the samples at 80 C until further analysis by SDS-PAGE and western blot. Transfer 200 l of highly purified mitochondria into a 1.5 ml pre-chilled microcentrifuge tube. Dilute mitochondria one-fold with ice-cold SEM buffer.
Using a sonicator compatible with small volumes, sonicate mitochondria 3 times for 30 seconds on ice. Centrifuge the sample for 30 minutes at 100, 000 x g at 4 C.And collect the supernatant in a new 1.5 ml pre-chilled microcentrifuge tube. After labeling the tube as S"for soluble protein fraction, keep the tube on ice.
Resuspend the pellet from the previous step in 400 l of ice-cold SEM buffer. Transfer 100 l of the resuspended pellet into a new 1.5 ml pre-chilled microcentrifuge tube. Label the tube as SMP"for submitochondrial particles fraction, and keep the tube on ice.
Dilute the remaining 300 l of resuspended pellet one-fold with freshly prepared 200 mM sodium carbonate. And incubate the diluted sample on ice for 30 minutes. Then, centrifuge the sample for 30 minutes at 100, 000 x g at 4 C.Collect the supernatant into a new 1.5 ml pre-chilled microcentrifuge tube.
Label the sample as CS"for carbonate supernatant fraction, and keep the tube on ice. Resuspend the pellet from the previous step in 400 l of ice-cold SEM buffer. And name the sample as CP"for carbonate precipitated fraction.
Precipitate all the samples with trichloroacetic acid to a final concentration of 10%and incubate the tubes on ice for 10 minutes. Then, centrifuge the samples for 10 minutes at 12, 000 x g at 4 C.After removing the supernatant, resuspend each pellet in the sample buffer. If the sample buffer becomes yellow, add 1 to 5 l of 1 M Tris base until it turns blue.
Add 1 l of 200 mM PMSF to all the tubes and store at 80 C until further analysis by SDS-PAGE and western blot. The crude mitochondrial fraction was purified on sucrose density gradient centrifugation, and reduction in other cellular contaminants was observed. Mitochondria to mitoplast conversion were monitored by the disappearance of the intermembrane space protein cytochrome b2 in the pellet.
With the concomitant appearance in the supernatant. The proteinase K mediated degradation of the intermembrane protein Sco1 was observed due to outer membrane disruption by osmotic shock. The outer mitochondrial membrane integrity was confirmed by the protection of cytochrome b2 and Sco1 against proteinase K degradation in the pellet fraction.
Similarly, the protection of the matrix soluble protein KGD confirmed the inner mitochondrial membrane integrity. Prx1 western blot profile suggested dual mitochondrial localization in intermembrane space and matrix. Mitochondria were subjected to sonication and carbonate extraction to investigate the topology of membrane proteins.
The integral membrane protein pouring remained in the pellet fractions. The KGD was detected in the supernatant fraction. The detection of KGD protein in the pellet was due to sonication parameter variations affecting the formation of SMPs.
After sodium carbonate treatment, the KGD and the SMP fraction was solubilized and found in the supernatant fraction. Prx1 western blot profile suggested an association with membrane periphery. For the success of the submitochondrial fractionation protocol, it is important to stop proteinase K activity after hypotonic swelling by adding PMSF to the samples.
Sites provide information about the submitochondrial protein localization. This protocol can also be used to check the integrity of mitochondrial preparations, which is fundamental during proteomic studies of mitochondrial subcompartments.
