Peptide-based interactions with calnexin target misassembled membrane proteins into endoplasmic reticulum-derived multilamellar bodies.
Oligomeric assembly of neurotransmitter transporters is a prerequisite for their export from the endoplasmic reticulum (ER) and their subsequent delivery to the neuronal synapse. We previously identified mutations, e.g., in the gamma-aminobutyric acid (GABA) transporter-1 (GAT1), which disrupted assembly and caused retention of the transporter in the ER. Using one representative mutant, GAT1-E101D, we showed here that ER retention was due to association of the transporter with the ER chaperone calnexin: interaction with calnexin led to accumulation of GAT1 in concentric bodies corresponding to previously described multilamellar ER-derived structures. The transmembrane domain of calnexin was necessary and sufficient to direct the protein into these concentric bodies. Both yellow fluorescent protein-tagged versions of wild-type GAT1 and of the GAT1-E101D mutant remained in disperse (i.e., non-aggregated) form in these concentric bodies, because fluorescence recovered rapidly (t(1/2) approximately 500 ms) upon photobleaching. Fluorescence energy resonance transfer microscopy was employed to visualize a tight interaction of GAT1-E101D with calnexin. Recognition by calnexin occurred largely in a glycan-independent manner and, at least in part, at the level of the transmembrane domain. Our findings are consistent with a model in which the transmembrane segment of calnexin participates in chaperoning the inter- and intramolecular arrangement of hydrophobic segment in oligomeric proteins.
Korkhov
Vladimir M
VM
Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University Vienna, Waehringer Strasse 13a, A-1090 Vienna, Austria.
Milan-Lobo
Laura
L
Zuber
Benoît
B
Farhan
Hesso
H
Schmid
Johannes A
JA
Freissmuth
Michael
M
Sitte
Harald H
HH
D002118:Calcium / Q000378:metabolism; D037281:Calnexin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D005959:Glucosidases / Q000037:antagonists & inhibitors; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D014759:Viral Envelope Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D050485:GABA Plasma Membrane Transport Proteins; D037102:Lectins; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D010455:Peptides; C493413:SLC6A1 protein, human; D014759:Viral Envelope Proteins; D037281:Calnexin; D005959:Glucosidases; D002118:Calcium