14-3-3 dimers probe the assembly status of multimeric membrane proteins

Background: Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (K-ATP) or GABA(B), G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For K-ATP channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown. Results: By employing a tetrameric affinity construct of the C terminus of the K-ATP channel alpha subunit, Kir6.2, we found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site. Conclusions: The COP[ vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. We propose a mechanism in which 14-3-3 bound to the correctly assembled multimer mediates release of the complex from the ER.