I have a protein that seems to relocate from the cell surface to an intracellular compartment when phosphorylated. When not phosphorylated the same protein appears to be associated to the cell membrane or outermost plasma membrane.
A classical membrane fractionation western blot experiment shows that this protein is always associated with the membrane pellet and not with the cytosolic fraction. The fraction markers are working perfectly.
I think that fusion to the outer membrane results in filopodial extensions that are enriched with my protein.
The attached image shows the distribution of a GFP fusion of my protein in 2 different conditions.
What should I do to try to test or demonstrate my idea that this protein is associated with membrane regions that can endocytose and become intracellular or can fuse with the surface membrane?
There is no transmembrane region.
Hello Neal,
You might find tips looking at the GPCR literature. G protein-coupled receptors are actually doing this in response to prolonged activation, called internalization. The receptors from the plasma membrane get internalized to endocytotic vesicles, later can be fused back to the plasma membrane in the original orientation, which process can be monitored with a labeled antibody against an extracellular epitope. You might get some tips from those experiments.
Besides the UC fractionation, you might track the route of your protein by co-labeling with different endocytotic marker proteins, there's a lot for early and late endosomes as well.
Best, Karoly
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