Scavenging of 14-3-3 proteins reveals their involvement in the cell-surface transport of ATP-sensitive K+ channels.
Arginine (Arg)-based endoplasmic reticulum (ER)-localization signals are involved in the quality control of different heteromultimeric membrane protein complexes. ATP-sensitive potassium (KATP) channels are unique because each subunit in the heterooctamer contains an Arg-based ER-localization signal. We have dissected the inactivation events that override the ER-localization activity of the eight peptide-sorting motifs. Employing a 14-3-3-scavenger construct to lower the availability of 14-3-3 proteins, we found that 14-3-3 proteins promote the cell-surface expression of heterologously expressed and native KATP channels. 14-3-3 proteins were detected in physical association with KATP channels in a pancreatic beta-cell line. Our results suggest that the Arg-based signal present in Kir6.2 is sterically masked by the SUR1 subunit. By contrast, 14-3-3 proteins functionally antagonized the Arg-based signal present in SUR1. The last ten amino acids were required for efficient 14-3-3 recruitment to multimeric forms of the Kir6.2 C-terminus. Channels containing a pore-forming subunit lacking these residues reached the cell surface inefficiently but were functionally indistinguishable from channels formed by the full-length subunits. In conclusion, 14-3-3 proteins promote the cell-surface transport of correctly assembled complexes but do not regulate the activity of KATP channels at the cell surface.
Heusser
Katja
K
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Yuan
Hebao
H
Neagoe
Ioana
I
Tarasov
Andrei I
AI
Ashcroft
Frances M
FM
Schwappach
Blanche
B
D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014981:Xenopus; D000099161:Kcnj11 Channel
D048948:14-3-3 Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; D008565:Membrane Proteins; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D000099161:Kcnj11 Channel