GFP in the lumen of the secretory pathway

Christian Stegmann c.stegmann at stud.uni-goe.de
Fri Feb 4 15:48:20 EST 2005


Austin P. So (Hae Jin) schrieb:
> nothwehrs at missouri.edu wrote:
> 
>> My
>> question relates to problems we have had adding GFP as a tag to a type
>> II membrane protein on the C-terminal end (lumenal side of the
>> membrane).  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.
> 
> 
> GFP is a heme protein with I forget which metal sitting in the middle of 
> the heme group. I am also unsure whether it is covalently linked to the 
> protein.

Wrong. The fluorophore originates from an internal Ser-Tyr-Gly sequence
which is post-translationally modified to an extended aromatic system.
The structure was solved in 1996.

To the original question: The folding environment in the ER is quite
different from that of the cytosol, thats for sure. In this case even
more important: The ER does not provide a reducing environment (remember
disulfides?), so my guess would be that the reduction of the C - C bond
whic is coupled with the cyclization of the neighboring glycine and
serine residues, is impeded. ==> No fluorescence.

But if have heard of another fluorophore that might work, i will try to
find that.

regards
christian





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