Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.
Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP(C)), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP(91). Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.
Domingo
Beatriz
B
Centro Regional de Investigaciones Biomédicas y Facultad de Medicina, Universidad de Castilla-La Mancha, 02006 Albacete, Spain.
Gasset
María
M
Durán-Prado
Mario
M
Castaño
Justo P
JP
Serrano
Antonio
A
Fischer
Thierry
T
Llopis
Juan
J
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002477:Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000473:pathology; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D014162:Transfection
D008565:Membrane Proteins; D011328:Prions; D049452:Green Fluorescent Proteins
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