Subunit counting in membrane-bound proteins.
The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.
Ulbrich
Maximilian H
MH
Department of Molecular and Cell Biology, University of California, Berkeley Material Science & Physical Bioscience Divisions, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Isacoff
Ehud Y
EY
D000818:Animals; D015220:Calcium Channels / Q000737:chemistry; D002478:Cells, Cultured; D003001:Cloning, Molecular; D054815:Cyclic Nucleotide-Gated Cation Channels; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry*; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D014982:Xenopus laevis
D015220:Calcium Channels; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D008565:Membrane Proteins; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins