We thank Dr. A. Tzagoloff (Columbia University) for providing antibodies raised against submitochondrial marker proteins Cyt. b2, &#945;KGD, and Sco1. We also thank Dr. Mario Henrique de Barros (Universidade de S&#227;o Paulo) for helpful discussion and comments during the establishment of this protocol.
This work was supported by research grants from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP) (grant 2013/07937-8).
Fernando Gomes and Helena Turano are also supported by FAPESP, grants 2017/09443-3 and 2017/23839-7, respectively. Ang&#233;lica Ramos is also supported by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES).

1. Growth of yeast cells
<ol>
	<li>Isolate single colonies of the strain of interest by streaking a small portion of the cells from a -80 &#176;C glycerol stock onto a YPD (1% yeast extract, 2% peptone, 2% glucose) agar plate. Incubate the plate at 30 &#176;C for 2-3 days. 
	NOTE: The S. cerevisiae strain used in this protocol is derived from BY4741 (MAT&#945;; his3&#916;1; leu2&#916;0; met15&#916;0; ura3&#916;0). With the ex...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intracellular+membranes+by+means+of+sodium+carbonate+treatment:+Application+to+endoplasmic+reticulum.">Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum.</a> Journal of Cell Biology. 93 (1), 97-102 (1982).</li><li>Glick, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+c1+and+b2+are+sorted+to+the+intermembrane+space+of+yeast+mitochondria+by+a+stop-transfer+mechanism.">Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.</a> Cell. 69 (5), 809-822 (1992).</li><li>Diekert, K., de Kroon, A. I., Kispal, G., Lill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+the+yeast+Saccharomyces+cerevisiae.">Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.</a> Methods in Cell Biology. 65, 37-51 (2001).</li><li>Glick, B. 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The authors declare no conflicts of interest.

The protocol presented here has been successfully used and continuously optimized for a long-time to determine the protein localization in the submitochondrial compartments13,14,18,21,22,23. The reliability and reproducibility of this protocol are strongly dependent on the purity and integrity of mitochondrial preparations<su...

Mitochondria are essential organelles of eukaryotic cells that play crucial roles in bioenergetics, cellular metabolism, and signaling pathways1. To properly execute these tasks, mitochondria rely on a unique set of proteins and lipids responsible for their structure and function. The budding yeast Saccharomyces cerevisiae has been widely used as a model system for investigations on mitochondrial processes, as well as for other organelles2. The mitochondrial genome codes for only eight proteins in yeast; the vast majority of mitochondrial proteins (~99%) are encoded by nuclear genes, which are translated on cytosolic ribosomes, and subsequently imported into their correct submitochondrial compartments by sophisticated protein import machineries3,4,5. Thus, mitochondrial biogenesis depends on the coordinated expression of both the nuclear and mitochondrial genomes6,7. Genetic mutations causing defects in mitochondrial biogenesis are associated with human diseases8,9,10.
In the past two decades, high-throughput proteomic studies targeting highly-purified mitochondria resulted in a comprehensive characterization of yeast mitochondrial proteome, which has been estimated&#160;to be composed of at least 900 proteins11,12,13,14. Although these studies provided valuable information, the suborganellar localization of each protein in the four mitochondrial subcompartments, namely, the outer membrane (OM), intermembrane space (IMS), inner membrane (IM), and matrix, is still required. This question was partially addressed with proteomic-wide studies of the two smaller mitochondrial subcompartments (OM and IMS)15,16. More recently, V&#246;gtle and collaborators made a major step forward by generating a high-quality global map of submitochondrial protein distribution in yeast. Using an integrated approach combining SILAC-based quantitative mass spectrometry, different submitochondrial fractionation protocols, and the data set from the OM and IMS proteomes, the authors assigned 818 proteins into the four mitochondrial subcompartments13.
Despite the advances achieved by these high-throughput proteomic studies, our knowledge about the submitochondrial proteome composition is far from being complete. Indeed, among 986 proteins reported by V&#246;gtle and collaborators as being localized into yeast mitochondria, 168 could not be assigned in any of the four submitochondrial compartments13. Moreover, the authors did not provide information about the membrane topology of proteins that were predicted to be peripherally attached to the periphery of mitochondrial membranes. For example, it is not possible to know if a protein that was assigned as peripherally attached to the inner membrane is facing the matrix or the intermembrane space. Apart from these missing data from the proteome-wide studies, there are conflicting information about the suborganellar localization of a significant number of mitochondrial proteins. One example is the protease Prd1, which has been assigned as an intermembrane space protein in the common databases such as Saccharomyces Genome Database (SGD) and Uniprot. Surprisingly, using a subfractionation protocol similar to that described here, V&#246;gtle and collaborators clearly showed that Prd1 is a genuine matrix protein13. As mentioned above, the submitochondrial localization of many mitochondrial proteins needs to be elucidated or reevaluated. Here, we provide a simple and reliable protocol to determine the suborganellar localization of yeast mitochondrial proteins. This protocol was developed and optimized by various research groups and has been routinely used to determine the submitochondrial localization, as well as the membrane topology of many mitochondrial proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Ultra-Clear Centrifuge Tubes, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA fatty acid free)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>Component of Homogenization buffer</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td>Component of DDT buffer</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td>Used to inactivate proteinase K</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Sigma-Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase-20T from Arthrobacter luteus</td>
			<td>MP Biomedicals, Irvine, CA</td>
			<td>320921</td>
			<td>Used to lyse living yeast cell walls to produce spheroplast</td>
		</tr>
	</tbody>
</table>

assessment of submitochondrial protein localization in budding yeast saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

The success of submitochondrial fractionation protocol depends on obtaining highly purified intact mitochondria. For this, it is essential that during the yeast cell lysis, the intactness of the organelles remains almost totally preserved. This is achieved by using a cell lysis protocol that combines the enzymatic digestion of the cell wall followed by physical disruption of the plasma membrane by using a Dounce homogenizer. The mitochondrial contents are then collected by differential centrifugation. This subcellular fr...

Watch this Scientific Journal Video about Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae at JoVE.com

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 functions.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

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.

assessment-submitochondrial-protein-localization-budding-yeast

Research

JoVE Journal

Biochemistry

2.7K visualizações.  Universidade de São Paulo. Este protocolo é projetado para determinar a localização subocondrial das proteínas mitocondriais de levedura, que é considerada como um passo fundamental durante a elucidação da função proteica mitocondrial. O protocolo é adequado para nossas cepas de levedura mantidas em diferentes condições de crescimento. Representando uma ferramenta poderosa para a pesquisa como o estudo das mitocôndrias.Para começar, células de raia de estoque de glicerol armazenadas a 80 C, em uma...