Recently we constructed a GFP (S65T) fusion to a membrane protein in yeast.
By biochemical experiments we concluded that the fusion was localized to
the vacuole, the yeast equivalent of the lysosome. The GFP fusion is
intact when localized to this organelle as shown by western blotting.
However, we could not detect any GFP flourescence using an appropriately
equipped 'scope that has successfully detected GFP fluorescence in the
past. The fusion was constructed such that GFP was on the lumenal side of
the membrane. Another group tagged the same protein in yeast with GFP
(S65T) on the cytosolic side and got a beautiful signal. The expression
levels between our experiment and theirs shouldn't have been dramatically
different. For various reasons it would be advantagous for us to have the
tag on the lumenal side.
My question is whether their should be any reason in principle why GFP in
the lumen of secretory or endosomal pathway organelles should give a poor
signal. I know that there is some affect of pH on the emission of GFP. But
the vacuole should be ~ pH 6.1 which is not low enough to cause a large
decrease in emission from what I have read. Has anyone had success
expressing GFP on the lumenal side and/or know of any reason as to why this
wouldn't work? Thanks.
--
Steve Nothwehr
401 Tucker
Division of Biological Sciences
Univ. of Missouri
Columbia, MO 65211
