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.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

BACKGROUND Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. RESULTS Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. CONCLUSION Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.