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Date: | Sun, 6 Apr 2014 14:07:45 -0500 |
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Dear Steve,
There are a few details that are missing to fully understand the issue. You
mention that the lipidation motif is from Ras, but which isoform of Ras? I guess
that it is the C-terminus of KRas, which is widely used. This motif carries a lot of
basic residues and is highly positively charged. It therefore interacts with
negatively charged phospholipids at the plasma membrane (PI(4,5)P2, PI(4)P
and PA), which is good for plasma membrane localization. However, these lipids
are essential modulators of various processes, e.g. interacting directly or
indirectly with the cytoskeleton
(http://www.ncbi.nlm.nih.gov/pubmed/24534649).
Another issue is the probe. GFP variants, certainly YFP, can dimerize when it is
concentrated at the surface of the membrane
(http://www.ncbi.nlm.nih.gov/pubmed/11988576). So do you know whether you
are using monomeric YFP variants (e.g. carrying the A206K mutation)? If these
proteins are of the 206A type, dimerization may increase PM interaction (good
for localization), thereby competing more strongly with endogenous proteins
depend on interaction with the PM via negatively charged lipids.
We have some experience with the lipidation motif of Lck which gives very good
PM labeling, still it does not seem to interfere with cell morphology. The Lck
motif was successfully used in fusion proteins with mTurquoise2 and mVenus
(the aa sequence preceding the CDS of the FP we used is MGCVCSSNPELPVAT).
However, our expts are mainly performed in HeLa cells, so it might be different
in neuronal cells.
Best Regards,
Joachim Goedhart
Section Molecular Cytology
SILS - University of Amsterdam
The Netherlands
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