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GFP in the lumen of the secretory pathway

Steve Nothwehr nothwehrs at missouri.edu
Thu Jan 6 18:46:50 EST 2005


I have a technical question about using GFP in yeast.  So far I
haven't found any good information in the literature about this but it
may be out there.  My question relates to problems we have had adding
GFP as a tag to a type II membrane protein on the C-terminal side
(lumenal side).  This single TMD protein is a resident of the late
Golgi/endosomal system of yeast.  When we add the GFP the protein is
made and is stable as shown by western blot and it appears to exit
from the ER. However, we see no fluorescence.  When we add the tag to
the N-terminus we get good fluorescence but then the localization of
the protein is perturbed somewhat.  We would really like to the get a
tag to work on the lumenal side.  We think the problem is related to
folding because when the tag is added to the N-terminus we sometimes
see staining in the lumen of vacuole when we introduce mutations that
affect retention in the Golgi.  So we think that if the GFP folds in
the cytosol and later the entire protein ends up in the vacuolar lumen
the GFP gives off decent fluorescence but if the folding occurs in the
ER lumen there is a problem.  Maybe someone has developed a variant of
GFP that is optimized for the lumen of the secretory pathway?  Of
course it is also possible that this is a problem related to our
protein and that if GFP were put on the C-terminus of another type II
protein it would give a good fluorescent signal.  I would REALLY
appreciate insights you have.  Thanks a lot.

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