TY - JOUR
T1 - Function recovery after chemobleaching (FRAC)
T2 - Evidence for activity silent membrane receptors on cell surface
AU - Sun, Haiyan
AU - Shikano, Sojin
AU - Xiong, Qiaojie
AU - Li, Min
PY - 2004/11/30
Y1 - 2004/11/30
N2 - Membrane proteins represent ≈30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression. We report the development of a pulse-chase assay to monitor function recovery after chemobleaching (FRAC) to probe the transit time of the Kir2.1 K+ channel to reach the cell surface. Our results reveal that the channel activity is contributed by a small fraction of channel protein, providing evidence of activity-silent "sleeping" molecules on the cell surface. This method distinguishes molecular density from functional density, and the assay strategy is generally applicable to other membrane receptors. The ability of the reported method to access the biogenesis pathways in a high-throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking.
AB - Membrane proteins represent ≈30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression. We report the development of a pulse-chase assay to monitor function recovery after chemobleaching (FRAC) to probe the transit time of the Kir2.1 K+ channel to reach the cell surface. Our results reveal that the channel activity is contributed by a small fraction of channel protein, providing evidence of activity-silent "sleeping" molecules on the cell surface. This method distinguishes molecular density from functional density, and the assay strategy is generally applicable to other membrane receptors. The ability of the reported method to access the biogenesis pathways in a high-throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking.
KW - Covalent modification
KW - Fluorescence recovery after photobleaching
KW - Ion channels
KW - Signaling
KW - Trafficking
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U2 - 10.1073/pnas.0404178101
DO - 10.1073/pnas.0404178101
M3 - Article
C2 - 15548608
AN - SCOPUS:10044230383
SN - 0027-8424
VL - 101
SP - 16964
EP - 16969
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 48
ER -