Adding membrane signal to cytoplasmic protein

Kevin Pumiglia pumiglk at aa.wl.com
Thu Oct 27 08:20:21 EST 1994


In article <1994Oct25.210606.8762 at mbcf>, mhughes at mbcf.stjude.org wrote:

> In article <01HIIC3VA6HU0008KV at UNCVX1.OIT.UNC.EDU>, ORSULIC at UNCVX1.OIT.UNC.EDU writes:
> > I am working on structure/function analysis of Drosophila Armadillo protein.
> > The protein is about 105 kb, does not have a signal or transmembrane sequence,
> > and is mostly cytoplasmic. 
> > 
> > I would like to generate the form of the protein that would be tethered to the
> > membrane. Any suggestions, protocols or references of successful addition of a
> > myristilation or membrane signal to other proteins would be appreciated. 
> > 
> > 
> > Sandra Orsulic
> > University of North Carolina
> > 
> > orsulic at uncvx1.oit.unc.edu
> 
> Sandra
> 	You might want to try adding a ras-like CAAX sequence to the c-terminus
> of you protein.  I don't know if this has been done successfully before or not, 
> but I'm almost positive that this sequence alone comprizes the entire
> myristilation signal for ras and many other proteins, and may work for
> armadillo.  
> 	Good luck,
> 
> 	Mark Hughes
> 	St. Jude Children's Research Hospital
> 	MHUGHES at mbcf.stjude.org

This approach has been used successfully.  Several times in Ras related
studies and more recently two papers this spring (June issues of Science
and or Nature, I can't recall the exact rteferneces right now) this
approach was used to Target Raf-1, a cytoplasmic kinase, to the membrane. 
The C-terminal portion (last 20 amino acids) of the K-Ras molecule has both
a polybasic domain and a farnesylation motif(CAAX).  Both are required for
membrane targeting.   In H-Ras, the polybasic domain is replaced by a
myristilation sequence; again both are neccessary for full membrane
localization.



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