Peptide-Based Interactions with Calnexin Target Misassembled Membrane Proteins into Endoplasmic Reticulum-Derived Multilamellar Bodies

Abstract

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 γ-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 ~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.

Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)

“…This finding was consistent with the reduced surface expression and V max of glycine transport shown by this mutant (Fig. 4A,B,D), and suggests that the unglycosylated transporter does not easily pass CNX quality control, an ER check point for newly synthesized glycoproteins [39]. This longer lasting binding may reflect an alteration to the 3-dimensional structure of the mutant that is detected by CNX.…”

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease

“…It is therefore logical that calnexin assesses the state of folding of PMP22 based on the accessibility of this TM segment. The recognition of dissociated helices within multispan membrane proteins by calnexin appears to be one of the mechanisms used by ER quality control to recognize membrane proteins that are incompletely folded or misfolded (Korkhov et al, 2008; Li et al, 2010; Swanton et al, 2003). Our observation regarding TM1 dissociation may explain why both WT and TrJ PMP22 navigate ER quality control to fold and traffic beyond the ER with only a low efficiency in vivo .…”

Psychiatric Drugs Bind to Classical Targets Within Early Exocytotic Pathways: Therapeutic Effects

“…This binding facilitates both intramolecular and intermolecular arrangements of hydrophobic segments (65,66). Presumably, like most molecules that exit the ER, the transporter must be correctly folded before binding to Sec24c or other members of the COPII cargo-binding protein Sec24 family (67).…”