I am expressing a plant ATPase transporter using pYES2 expression vector into saccharomyces cerevisiae. How may i know the location of the transporter expressed within the yeast cell ? Is it going to be at the same location like in plant cell (eg : plasma membrane) or else ? Please suggest some paper to refer to.. THANKS A BUNCH !!
Well, I would suggest to try to localize your protein. For a rough estimation you can perform a sucrose gradient fractionation of the microsomal membrane fraction obtained from your yeast cells. You can start here: http://www.med.unc.edu/~dohlmahg//sucrose.html After the sucrose density gradient centrifugation you can perform SDS-PAGE + Wester blotting with the different fractions and find your protein in one (or a few) membrane fractions.
Hi Alajos and Lucia,
Thanks for suggestion. Really appreciate it! I will go for tagging the gene with fluorescent protein and do the subcellular localization study. Just came across an article by courbot 2007. Cant do the western blot as mine is lacking of the His / Strep tagg. Thanks anyways !!
Hi Zakuan,
You do not need to wait until you get the tagged protein expressed; since you have a plant ATPase for finding in the different yeast membrane fractions you can run enzyme assay in the sucrose gradient fractions and localize your protein. I do believe you have special inhibitor for your plant ATPase; so you can distinguish between the different ATPases you will be able to detect in enzyme assays in the different membrane fractions (e.g. olygomycin-sensitive, nitrite-sensitive, vanadate-sensitive, etc.). Godd luck to the experiments.
Well, since yeast cells also have vanadate-sensitive ATPase, you would need a proper tag on your expressed ATPase for distinguishing it from the yeast one. It is just might be that your expressed vanadate-sensitive ATPase will be localized similarly to that of the yeast protein.
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