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

TY - JOUR

T1 - 14-3-3 dimers probe the assembly status of multimeric membrane proteins

AU - Yuan, Hebao

AU - Michelsen, Kai

AU - Schwappach, Blanche

PY - 2003/4/15

Y1 - 2003/4/15

N2 - 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 COPI 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.

AB - 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 COPI 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.

KW - 14-3-3 Proteins

KW - Arginine/metabolism

KW - Binding, Competitive/physiology

KW - Blotting, Western

KW - Endoplasmic Reticulum/physiology

KW - Gene Expression Profiling

KW - HeLa Cells

KW - Humans

KW - Membrane Proteins/metabolism

KW - Potassium Channels, Inwardly Rectifying/metabolism

KW - Precipitin Tests

KW - Signal Transduction/physiology

KW - Tyrosine 3-Monooxygenase/metabolism

U2 - 10.1016/s0960-9822(03)00208-2

DO - 10.1016/s0960-9822(03)00208-2

M3 - SCORING: Journal article

C2 - 12699619

VL - 13

SP - 638

EP - 646

JO - CURR BIOL

JF - CURR BIOL

SN - 0960-9822

IS - 8

ER -