Colleagues,
We study a yeast type II integral membrane (single trans-membrane
domain) protein that is a resident of the Golgi/endosomal system. We
have successfully tagged this protein on the N-terminus (cytosolic
side) with GFP and get a good fluorescent signal. However, for
technical reasons we really would like to add the GFP tag to the
C-terminus (lumenal side). We have done this and protein is expressed
and is stable. However, the problem is that there is NO fluorescence.
This leads me to believe that either GFP is not compatible with the
lumenal domain of our protein or that there is a general problem with
the folding of GFP in the lumen of the ER. By the way we have good
evidence that the C-terminally tagged protein exits from the ER. Does
anyone know of any variants of GFP that are known to function well in
the lumen of the secretory pathway?
I suspect 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 folding instead occurs in the ER lumen there is a
problem.
I would REALLY appreciate insights you have. Thanks a lot.
Steve
