see BBRC 250:335-341 (1998)
>To: nobody at net.bio.net>X-Really-To: yeast at net.bio.net>Newsgroups: bionet.molbio.yeast
>From: nothwehr at biosci.mbp.missouri.edu (NothwehrS)
>Subject: GFP in lumen of sec. pathway organelles
>Date: Fri, 11 Dec 1998 09:26:13 -0600
>Content-Length: 1394
>>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
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David Mueller
Department of Biochemistry and Molecular Biology
The Chicago Medical School
3333 Greenbay Rd.
North Chicago, IL
60064
Tel: 847-578-8606
FAX: 847-578-3240
