The Endoplasmic Reticulum Membrane Complex Promotes Proteostasis of GABA_AReceptors

Abstract

The endoplasmic reticulum membrane complex (EMC) plays a critical role in the biogenesis of tail-anchored and a subset of multi-pass membrane proteins in the endoplasmic reticulum. However, due to the nearly exclusive expression of neurotransmitter-gated ion channels in the central nervous system, the role of the EMC in their biogenesis is not well understood. In this study, we demonstrated that the EMC positively regulates the surface trafficking and thus function of endogenous γ-aminobutyric acid (GABAA) receptors, the primary inhibitory ion channels in the mammalian brain. Further, among ten EMC subunits, EMC3 and EMC6 have the most prominent effects, indicating a subunit-specific contribution. EMC3 and EMC6 show endogenous interactions with major neuroreceptors, which depends on their transmembrane domains. Overexpression of EMC3 and EMC6 is sufficient to restore the function of epilepsy-associated GABAA receptor variants, suggesting that operating EMC has the potential to ameliorate neurological diseases associated with protein conformational defects.In briefThe multi-subunit EMC serves as an insertase for a subset of membrane proteins and enables their biogenesis in the endoplasmic reticulum. However, the subunit-specific effect of the EMC on multi-pass neuroreceptors is not well understood. Whittsette et al. demonstrate that EMC3 and EMC6 interact with GABAA receptors and positively regulate their trafficking and function.HighlightsEMC3 and EMC6 positively regulate the function of endogenous GABAA receptors.The EMC interacts with major endogenous neuroreceptors.The interaction between EMC and GABAA receptors depends on the EMC transmembrane domains.Overexpressing the EMC is sufficient to restore the function of pathogenic GABAA receptor variants.

Quantitative interactome proteomics identifies proteostasis network for GABAAreceptors

“…GABA A receptor subunits are co-translationally targeted to the ER membrane ( Figure 5 , State 1), which is mediated by the signal recognition particle (SRP) complex (49). The insertion of the transmembrane domains of GABA A receptors into the lipid bilayer could be facilitated by the SEC61 translocon as well as the ER membrane complex (EMC) (50-52). Our SILAC analysis identified SRP68 and SSR1, which is the α subunit of a translocon-associated protein.…”

Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

“…Lentivirus production and transduction in neurons was performed as described previously [80]. Briefly, HEK293T cells were transfected with a Hsp47-set of four siRNA lentivectors (rat) (Applied Biological Materials, catalog #: 435050960395), or scrambled siRNA GFP lentivector (Applied Biological Materials, catalog #: LV015-G) together with psPAX2 and pMD2.G plasmids using TransIT-2020.…”

“…Neuron staining and confocal immunofluorescence microscopy analysis were performed as described previously [49, 79]. Briefly, to label cell surface proteins, primary neurons on coverslips were fixed with 2% paraformaldehyde in DPBS for 10 min.…”