In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation.
We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [(32)P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be approximately 1.6 times the rate of translation.
Goder
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V
Biozentrum, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Crottet
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P
Spiess
M
M
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D019556:COS Cells; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D037263:Asialoglycoprotein Receptor; D008565:Membrane Proteins; D009842:Oligopeptides; D010710:Phosphates; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases