Yeast Mito protein question?

fanwang at gpu.srv.ualberta.ca Yi
Mon Jul 10 22:11:20 EST 1995


Hi there! I am working on a yeast mitochondrial protein right now. The 
problem I am trying to solve is to demonstrate that "my protein"(about 
80 kDa) is truly mitochondrial protein. What I have demonstrated is 
this protein is found in the mitochondrial fraction only, not cytosol.
However, I could not make the in vitro import experiment to work to 
show the cleavage of the presequence (if exist?) which is typical for 
mitochondrial proteins. I am thinking about doing an experiment to show 
that the mature protein in isolated mitochondria is smaller in size on 
Western Blot than the precursor accumulated in cytosol by incubation in 
the presence of an uncoupler, CCCP. I have seen that CCCP is used to 
treat yeast culture for short period of time such as in the pulse/chase 
experiment but not during the culturing of yeast. So, my question is 
whether it is possible that yeast will grow in the presence of CCCP with 
glucose as carbon source, and therefore my protein (probably along with 
other mito proteins) will accumulated as precursor protein. Or at which 
point of yeast growth phase is CCCP added to get this protein accumulated 
to certain amount to be detected by Western Blot? If it is possible to do 
this experiment, I might save me some time to do pulse/chase and 
immunoprecipitation to demonstrate the same thing in shorter period of 
time. I hope all the experts out there could help me on this one. Any 
suggestion is welcome. Thank you all.

Yi 
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FAN WANG E-MAIL: FANWANG at GPU.SRV.UALBERTA.CA
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