The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors

Whittsette, Angela L.; Wang, Ya Juan; Mu, Ting

doi:10.1016/j.isci.2022.104754

Cited by 12 publications

(11 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…From this manually curated list, we chose for analysis six proteins containing terminal TMDs of varying hydrophobicity and C-tail lengths. The functional expression of five of these proteins, or their close homologs, have been observed in earlier studies to be dependent on EMC 26 , 27 , 31 , 33 . Therefore, we tested their terminal TMDs in the 23L-TMD reporter with a C-terminal glycosylation site (Extended Data Fig.…”

Section: Resultsmentioning

confidence: 63%

See 1 more Smart Citation

EMC rectifies the topology of multipass membrane proteins

Wu,

Smalinskaitė,

Hegde

2023

Nat Struct Mol Biol

View full text Add to dashboard Cite

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC’s cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome–translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

show abstract

Section: Resultsmentioning

confidence: 63%

“…To examine a potential role for EMC in Cys-loop channels, we focused on GABA A receptors, whose reliance on EMC has also been seen in mammals 27 . Using a previously characterized HEK293 cell line with stable inducible expression of a heteropentameric GABA A receptor 28 , 29 , we tested the effect of acute EMC depletion.…”

Section: Resultsmentioning

confidence: 99%

EMC rectifies the topology of multipass membrane proteins

Wu,

Smalinskaitė,

Hegde

2023

Nat Struct Mol Biol

View full text Add to dashboard Cite

show abstract

“…Whole-cell currents were recorded for HEK293T cells expressing GABA A receptors. Automated patch clamping was performed on the Ionflux Mercury 16 instrument (Fluxion Biosciences, California), as previously described 14 In the above equation, y is the normalized peak current, x is log(Concentration of GABA), x0 is the Half maximal effective concentration (EC 50 ), and p is the Hill coefficient.…”

Section: Automated Patch-clamping With Ionflux Mercury 16 Instrumentmentioning

confidence: 99%

“…Neuron staining and confocal immunofluorescence microscopy analysis were performed as described previously 14 . Briefly, to label cell surface proteins, iPSC-derived GABAergic neurons on the chamber slides were fixed with 4% paraformaldehyde in DPBS for 10 min.…”

Section: Immunofluorescence Staining and Confocal Microscopymentioning

confidence: 99%

“…Recently, our quantitative proteomics identified a proteostasis network for GABA A receptors, which enabled our efforts to further elucidate their biogenesis pathways 13 . Upon the synthesis of GABA A receptor subunits, the ER membrane protein complex (EMC), including EMC3 and EMC6, facilitates the insertion of the transmembrane domain of GABA A receptors into the ER membrane 14 . Then BiP (binding immunoglobulin protein) and calnexin in the ER act as profolding chaperones to promote the productive folding of GABA A receptors 15,16 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants

Wang

Seibert

Ahn

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the alpha1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the alpha1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the alpha1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner.

show abstract

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3

Li,

Shen,

Cheng

et al. 2023

Heliyon

View full text Add to dashboard Cite

The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors

Cited by 12 publications

References 68 publications

EMC rectifies the topology of multipass membrane proteins

EMC rectifies the topology of multipass membrane proteins

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3

Contact Info

Product

Resources

About

The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors

Cited by 12 publications

References 68 publications

EMC rectifies the topology of multipass membrane proteins

EMC rectifies the topology of multipass membrane proteins

Pharmacological chaperones restore proteostasis of epilepsy-associated GABAAreceptor variants

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3

Contact Info

Product

Resources

About

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants