Hi there,
I am trying to localize one of my plant test gene in yeast cells. I m using pDR196 yeast expression vector. I cloned GFP and test gene under PMA promoter (Yeast specific promoter). I prepared a suspesion of single positive colony of yeast in 15 microlitre of water and checked under confocal microscope using 63X and FITC. But signal was diffuse. It supposed to be on plasma membrane of yeast since the test gene localizes on plant plasma membrane. Can any one suggest any protocol to improve localization or induce the signal? or is there any way to induce PMA promoter? Did any one do these kind of experiment?
Please suggest.
Hi Megha,
I am thinking if the problem could be the lack of, or the position of the localization signal peptide.
To this end could you please send more details on the vector, including its basic structure, and the relative localization of the of the plant gene and the GFP reporter.
In this line, you could try putting the GFP on the other terminus to exclude masking of the signal peptide. Also, why do you go for expression in yeast? Have you tried expression of this construct (with a suitable promoter) in plant cells (e.g. protoplasts)?
Is your protein processed after targeting to the membrane? Could be that GFP is cleaved off and what you see is localization of free GFP (I'd suggest a western blot on the yeast cell samples for this with a GFP antibody and an antibody against your test gene).
Just want to add something to previous answers. Expressing plant proteins in yeast can result in some very unpredictable outcomes. Generally speaking, highly conserved proteins between plants and yeast tend to give the expected results as the function is also conserved which makes yeast a simple and convenient system to study those protein. Plant-specific proteins, in so many cases, do not work well in yeast and tend to show some very weird results. So in many cases yeast isn't really a good system to study plant proteins and in those cases it is better to avoid using yeast all together.
Assuming that the protein you're working on works in yeast exactly the same way it works in plants, then there are a couple things you can do to troubleshoot the problem.
1. Just to elaborate on Sascha's point, make sure that the GFP tag does not affect the function of the protein by expressing one version of the protein with c-terminal tag and another with N-terminal tag. Then follow what Sascha has suggested, doing western tests.
2. Make sure that the protein is functional in yeast cells, I'd highly recommend expressing the same tagged protein in plants cells or you can do quick transient expression in N. benthamiana or N. Tabacum beside yeast cells. This should give you a clear answer about wether the protein actually works in yeast or not.
Did you checked the expression of protein by Western blotting at least 3 to 4 independent yeast colonies with anti GFP the go for the microscopic examination of the positive constructs . Also GO FOR DNA sequencing.
And at least take more than one colony for screening. I am doing the same !
But my vector is customized according to my needs.
All the best 👍
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