ER membrane marker protein

[Fischi]

Hello!

 

I'm looking for a very special protein, I know how it should look like, but it's not easy to find:

 

- a transmembrane protein residing exclusively in the ER

- ideally Type I membrane protein with short or no lumenal part

- N-terminal region self-sufficient for insertion into membrane

 

Perhaps you can already imagine, what I'm trying to do. I need this protein to label the ER membrane with a fluorescent protein in the cytosol. (An antibody staining does not help me, then I would not post this here ) It should not interfere with any biological process at the ER membrane, that's why I don't like a functional lumenal part.

When anybody knows a paper, where they have done something similar... that would be great!

I'm searching pubmed for two days now and couldn't find anything suitable.

 

Thanks a lot!!!

[Max_Normal]

The standard transmembrane ER marker is Calnexin. It's a medium sized protein, and should be easy to clone if you are making a GFP chimera. Don't confuse this with Calregulin, which shares a lot of homology but is lumenal rather than transmembrane. The sequence will already have an SRP insertion sequence, I expect, so you should be OK with the GFP on the C-terminus.

 

If you just need to visualise the ER (although this will not be membrane associated, so presumably you don't, unless you just hate yourself), a good way is to simply add a KDEL sequence to the N-terminus of the fluorescent protein. KDEL-GFP will be continuously recycled through the ER and you will get a nice reticular stain. In fact I have one of these in RFP and one in GFP, contact me if you do want an aliquot, and i'll post it.

 

Alternatively you can use lipid based markers such as C5- and C6-ceramide-BODIPY, these will insert into the ER/golgi membrane (make sure you choose which one is enriched in the ER carefully).

 

Other than that, you could clone of the proteins that makes up the signal Recognition Particle that the nascent peptide is extruded into.

Edited March 3, 2012 by Max_Normal