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On isolating yeast mitochondria Print E-mail

On Isolating and testing mitochondria from yeast

  An excerpt from “Mitochondria Interest Group“ ( ) mail list.
2013-2014

 Download yeast_mito.pdf
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Q: (Dr. John J. Lemasters):

I seek advice on isolating mitochondria from yeast to determine basic bioenergetic parameters (state 3, state 4; ion uptake, etc.):
1. How specifically should the yeast be grown (fermentable or non-fermentable medium, etc.)?
2. What is a simple but good isolation procedure that can be performed by us vertebrate mitochondria folks?
3. What reaction medium is best for determining bioenergetic parameters? What respiratory substrate should be used?

A1: (Dr. J.Pedro F.Murray)

  Those are the standard protocols for yeast mitochondria isolation yeast has to be grown under respiring conditions (2% Na lactate, pH 5.6). Regarding conditions for bioenergetic experiments you have to seek help somewhere else, not my expertise.
http://www.ncbi.nlm.nih.gov/pubmed/6290489 [Daum G, Böhni PC, Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028-33.],
http://www.ncbi.nlm.nih.gov/pubmed/11381604 [Diekert K, de Kroon AI, Kispal G, Lill R. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 2001;65:37-51.]

A2: (Dr. Marco Colombini)

  The following link gives you a paper we published in 1998. The method describes in detail how to grow and isolate mitochondria from yeast. To obtain functional mitochondria with a mainly intact outer membrane they should be grown on lactate. https://dl.dropboxusercontent.com/u/15495627/JMB%201998.pdf [Lee AC, Xu X, Blachly-Dyson E, Forte M, Colombini M. The role of yeast VDAC genes on the permeability of the mitochondrial outer membrane. J Membr Biol. 1998 Jan 15;161(2):173-81. PubMed PMID: 9435273.]

A3: (modified from the post of Dr. Eduardo Rial)

  To give you an idea, from an 800 ml yeast culture grown to 4-5 OD, we routinely obtain close to 1 ml of a mitochondrial suspension at 20-30 mg/ml. Our control yeast mitochondria routinely show an RCR just above 3 (with 0.1 - 0.2 mM ADP). It is not like vertebrate mitochondria. To get a good yield the best for us has been the use of lactate as carbon source although we have also grown them in galactose. With respect to the substrate for respiration we routinely use NADH (1-3 mM). We have not published a detailed protocol for yeast mitochondria isolation but the procedure is based on the one originally developped by Guerin's group and it is based on the enzymatic digestion of the cell wall and the subsequent isolation of mitochondria by differential centrifugation. The entire procedure takes 3-4 hours

 

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