scholarly journals Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lakshmi E Miller-Vedam ◽  
Bastian Bräuning ◽  
Katerina D Popova ◽  
Nicole T Schirle Oakdale ◽  
Jessica L Bonnar ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Membrane Protein Complex ◽  
Functional Studies ◽  
Protein Levels ◽  
Protein Biogenesis ◽  
Mechanistic Basis ◽  
Specific Regulation ◽  
Protein Classes ◽  
Polytopic Membrane Protein ◽  
Lumenal Domain

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

scholarly journals Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

2020 ◽  
Author(s):  
Lakshmi E. Miller-Vedam ◽  
Bastian Bräuning ◽  
Katerina D. Popova ◽  
Nicole T. Schirle Oakdale ◽  
Jessica L. Bonnar ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Membrane Protein Complex ◽  
Functional Studies ◽  
Protein Levels ◽  
Protein Biogenesis ◽  
Mechanistic Basis ◽  
Specific Regulation ◽  
Protein Classes ◽  
Polytopic Membrane Protein ◽  
Lumenal Domain

AbstractMembrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies revealed at least two separable EMC activities, as an insertase regulating tail-anchored protein levels and as a polytopic membrane protein holdase chaperone. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a novel, non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

scholarly journals Structure of BamA, an essential factor in outer membrane protein biogenesis

2014 ◽  
Vol 70 (6) ◽  
pp. 1779-1789 ◽  
Author(s):  
Reinhard Albrecht ◽  
Monika Schütz ◽  
Philipp Oberhettinger ◽  
Michaela Faulstich ◽  
Ivan Bermejo ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Cell Envelope ◽  
Significant Degree ◽  
State Function ◽  
E Coli ◽  
Functional Studies ◽  
C Terminus ◽  
Protein Biogenesis

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. InEscherichia colithis function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of theE. coliBamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA fromHaemophilus ducreyiandNeisseria gonorrhoeaeand are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation,E. coliBamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer.E. coliBamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

scholarly journals Co-translational biogenesis of lipid droplet integral membrane proteins

2021 ◽  
Author(s):  
Pawel Leznicki ◽  
Hayden O. Schneider ◽  
Jada V. Harvey ◽  
Wei Q. Shi ◽  
Stephen High
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Neutral Lipids ◽  
Storage Site ◽  
Hairpin Loop ◽  
Membrane Protein Complex ◽  
Molecule Inhibitor ◽  
Protein Biogenesis ◽  
Intracellular Storage ◽  
Loop Conformation

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N-termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

scholarly journals The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ashley M Ngo ◽  
Matthew J Shurtleff ◽  
Katerina D Popova ◽  
Jessie Kulsuptrakul ◽  
Jonathan S Weissman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Transmembrane Protein ◽  
Point Mutations ◽  
Transmembrane Proteins ◽  
Physical Interaction ◽  
Translation Efficiency ◽  
Membrane Protein Complex ◽  
Protein Biogenesis ◽  
Er Membrane

Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential for accurate folding and post-translational stability rather than translation efficiency. Specifically, we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts an aberrant topology in the absence of the EMC leading to degradation. Together, our data highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing challenging transmembrane proteins during synthesis.

scholarly journals Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicholas J. Barrows ◽  
Yesseinia Anglero-Rodriguez ◽  
Byungil Kim ◽  
Sharon F. Jamison ◽  
Caroline Le Sommer ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Viral Entry ◽  
Protein Complex ◽  
Dual Roles ◽  
Membrane Protein Complex ◽  
Protein Biogenesis ◽  
Flavivirus Infection ◽  
Er Membrane

scholarly journals Regulated assembly of the ER membrane protein complex

2020 ◽  
Author(s):  
Tino Pleiner ◽  
Kurt Januszyk ◽  
Giovani Pinton Tomaleri ◽  
Robert S. Oania ◽  
Masami Hazu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Hydrophobic Surface ◽  
Amphipathic Helix ◽  
Membrane Protein Complex ◽  
Interaction Partners ◽  
Protein Biogenesis ◽  
Assembly Factor ◽  
Membrane Spanning ◽  
Er Membrane

SummaryThe assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome.

scholarly journals The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

2019 ◽  
Author(s):  
Ashley M Ngo ◽  
Matthew J Shurtleff ◽  
Katerina D Popova ◽  
Jessie Kulsuptrakul ◽  
Jonathan S Weissman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Transmembrane Protein ◽  
Point Mutations ◽  
Transmembrane Proteins ◽  
Physical Interaction ◽  
Translation Efficiency ◽  
Membrane Protein Complex ◽  
Protein Biogenesis ◽  
Er Membrane

AbstractFlaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential for accurate folding and post-translational stability rather than translation efficiency. Specifically, we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts an aberrant topology in the absence of the EMC leading to degradation. Together, our data highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing challenging transmembrane proteins during synthesis.

scholarly journals Co-translational biogenesis of lipid droplet integral membrane proteins

2021 ◽  
Author(s):  
Pawel Leznicki ◽  
Wei Q Shi ◽  
Stephen High
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Neutral Lipids ◽  
Storage Site ◽  
Hairpin Loop ◽  
Membrane Protein Complex ◽  
Molecule Inhibitor ◽  
Protein Biogenesis ◽  
Intracellular Storage ◽  
Loop Conformation

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. Strikingly, this route can also result in a transient exposure of the short N-termini of LD membrane proteins to the ER lumen, followed by topological rearrangements that enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

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