EGFP (F64L,S65T) is significantly more sensitive to acid pH than wtGFP, with
fluorescence dropping off below pH ~7.0. If I remember correctly, S65T is
similar in this regard to EGFP. I therefore suspect that pH sensitivity could be
the reason you don't observe fluorescence.
I also remember that GFPuv (a.k.a. cycle 3) is like wtGFP in being less
sensitive to acid pH. Since GFPuv gives a much stronger fluorescence than wtGFP
this variant may work for your application.
Both David Piston's lab and William Ward's lab have looked at the pH sensitivity
of GFP variants so look for their publications if you want to check my
recollections.
-=Paul=-
""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
" Paul Kitts, Ph.D. NCBI/NLM/NIH "
" Email: kitts at ncbi.nlm.nih.gov Bldg 38A, 8N803 "
" Phone: 301 435-6091 8600 Rockville Pike "
" Fax: 301 480-9241 Bethesda, MD 20894, U.S.A. "
""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
To: fluorpro at net.bio.net
From: nothwehr at biosci.mbp.missouri.edu (NothwehrS)
Subject: GFP in lumen of sec. pathway organelles
Date: Fri, 11 Dec 1998 09:10:04 -0600
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
