Short answer, yes. They're relatively delicate organelles, and freeze/thaw cycles will likely rupture them at least slightly. Further, you'll be stressing the yeast considerably so will also be getting stress response alterations.

Plus of course isolating functional mitos from yeast is fairly non-trivial anyway (pesky cell walls) so I would tend to do everything as fresh as possible.

How are you isolating them, and what functional assays are you doing? It might be possible to just do stuff in situ with permeabilised yeast cells and skip isolation altogether.

Freezing and thawing will seriously affect the oxidative phosphorylation or any other function, which needs an intact electron transport chain. However, if your interest is only to measure a soluble enzyme activity it may not matter. 

Please note that the native mitochondrial morphology is lost when you isolate them. Isolated mitochondria are always spherical because the attachment to the cytoskeleton is lost. However, it is possible to perform ultrastructural studies with isolated mitochondria. Don't use previously frozen mitochondria for this analysis because artefacts will most likely be present which make interpretation of data difficult or misleading.

Cheers, Christian

hi 

Thanks John, Pothana and Christian for your replies

Actually I am isolating mitochondria to study membrane polarisation as a function of mitochondrial health after giving it a treatment that is supposed to make the mitochondria sick, as it is evident by fragmented mitochondria under microscope after treatment . I believe that if mitochondria is sick, membrane should depolarise, however, even after chronic treatment, membrane is hyperpolarised. (although this is an entirely different question which If any of you can answer, it would help me)

Further, continuing with the queries of current question

1. I am isolating mitos using a protocol standardised in my PI's lab that includes using Tris so4 DTTbuffer, 1.2M Sorbitol buffer and 0.6M sorbitol homogenisation buffer

further storing it in 0.6Msorbitol hepes buffer

2. I am, as per treatment to my cells, isolating mitos from cells of 0.6-0.8 od/ml

3. I have to check membrane polarisation using these mitos.

Also please note that I can not do the whole process including prep and assay in one day as I have to induce the culture for three hours first and then mito prep itself needs 2 hrs incubation for spheroplasting.

I have several options

1. I do overnight secondary culture and induce cells in morning and store mitos at -80 for further assays

2. I do secondary culture in the day (doubling time of my strain is 3hrs in YPR), induce it for three more hours, pellet it and do mito prep next day and continue with functional assay

3. I prepare spheroplasts and store it at 4degree and do assay next day using membrane permeable dye

looking forward to your help in the topic

Pratima 

Hello Pratima,

unfortunately none of the options you propose is suitable for conducting your experiments. The reason is that in all cases you will heavily stress the cells which in turn affects mitochondrial integrity. You need to cultivate the cells, isolate the mitochondria and then immeaditely perform your measurements. Maybe somebody else in the lab could help you with the experiment (e. g., starting early, preparing spheroplasts and then you continue with the assays).

Best regards, Christian

Dear Pratima,

Some own practical dealing with mitochondria in cell cultures might help in your research. Recent Canadian studies (2014) repeated our observations in the 70s that cellular function and viability of high-energy demanding cells traditionally viewed as relied on the continuous supply of ATP, and that utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. Instead, it was shown that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. The above prevented mitochondrial mitochondrial dysfunction, fragmentation and cell death. The findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism.

Best wishes

Ilya