scholarly journals The ER transmembrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

2021 ◽  
Author(s):  
Büsra Güngör ◽  
Tamara Flohr ◽  
Sriram G Garg ◽  
Johannes M. Herrmann
Keyword(s):  
Protein Complexes ◽  
Insertion Reaction ◽  
Membrane Insertion ◽  
Deletion Mutants ◽  
Hydrophobic Core ◽  
Protein Insertion ◽  
Membrane Complex ◽  
Membrane Protein Insertion ◽  
Insertion Mechanism ◽  
Get Complex

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

scholarly journals A Continuum Method for Determining Membrane Protein Insertion Energies and the Problem of Charged Residues

2008 ◽  
Vol 131 (6) ◽  
pp. 563-573 ◽  
Author(s):  
Seungho Choe ◽  
Karen A. Hecht ◽  
Michael Grabe
Keyword(s):  
Membrane Protein ◽  
Protein Complexes ◽  
Computational Cost ◽  
Atomistic Simulations ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Membrane Protein Complexes ◽  
Dynamics Simulations ◽  
Charged Peptides ◽  
Continuum Theories

Continuum electrostatic approaches have been extremely successful at describing the charged nature of soluble proteins and how they interact with binding partners. However, it is unclear whether continuum methods can be used to quantitatively understand the energetics of membrane protein insertion and stability. Recent translation experiments suggest that the energy required to insert charged peptides into membranes is much smaller than predicted by present continuum theories. Atomistic simulations have pointed to bilayer inhomogeneity and membrane deformation around buried charged groups as two critical features that are neglected in simpler models. Here, we develop a fully continuum method that circumvents both of these shortcomings by using elasticity theory to determine the shape of the deformed membrane and then subsequently uses this shape to carry out continuum electrostatics calculations. Our method does an excellent job of quantitatively matching results from detailed molecular dynamics simulations at a tiny fraction of the computational cost. We expect that this method will be ideal for studying large membrane protein complexes.

scholarly journals The architecture of EMC reveals a path for membrane protein insertion

2020 ◽  
Author(s):  
John P. O’Donnell ◽  
Ben P. Phillips ◽  
Yuichi Yagita ◽  
Szymon Juszkiewicz ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Integral Membrane Proteins ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Chaperone Function ◽  
Eukaryotic Genes ◽  
Biochemical Assays ◽  
Protein Biogenesis ◽  
Membrane Protein Insertion

AbstractApproximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function.

scholarly journals The architecture of EMC reveals a path for membrane protein insertion

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
John P O'Donnell ◽  
Ben P Phillips ◽  
Yuichi Yagita ◽  
Szymon Juszkiewicz ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Integral Membrane Proteins ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Chaperone Function ◽  
Eukaryotic Genes ◽  
Biochemical Assays ◽  
Protein Biogenesis ◽  
Membrane Protein Insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function.

scholarly journals Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG–SecDF–YajC–YidC

2016 ◽  
Vol 473 (19) ◽  
pp. 3341-3354 ◽  
Author(s):  
Joanna Komar ◽  
Sara Alvira ◽  
Ryan J. Schulze ◽  
Remy Martin ◽  
Jelger A. Lycklama a Nijeholt ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Signal Recognition Particle ◽  
Common Mechanism ◽  
Membrane Insertion ◽  
Transmembrane Helices ◽  
Protein Insertion ◽  
Wide Range ◽  
Membrane Protein Insertion ◽  
Bona Fide ◽  
Lateral Gate

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

scholarly journals Isolation of Cold-Sensitive yidC Mutants Provides Insights into the Substrate Profile of the YidC Insertase and the Importance of Transmembrane 3 in YidC Function

2007 ◽  
Vol 189 (24) ◽  
pp. 8961-8972 ◽  
Author(s):  
Jijun Yuan ◽  
Gregory J. Phillips ◽  
Ross E. Dalbey
Keyword(s):  
Membrane Protein ◽  
Expression System ◽  
Point Mutations ◽  
Integral Membrane Protein ◽  
Cold Sensitivity ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Amino Terminal ◽  
Membrane Protein Insertion ◽  
Cold Sensitive

ABSTRACT YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the yidC(C423R) mutant confers a weak phenotype on membrane protein insertion while a yidC(P431L) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F1Fo subunit c (FoC), while CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that −3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while FoC required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.

scholarly journals BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly

2017 ◽  
Vol 474 (23) ◽  
pp. 3951-3961 ◽  
Author(s):  
Yinghong Gu ◽  
Yi Zeng ◽  
Zhongshan Wang ◽  
Changjiang Dong
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Insertion ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Protein Insertion ◽  
E Coli ◽  
Functional Studies ◽  
Functional Assays ◽  
Membrane Protein Insertion

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a ‘top hat’-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C–β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C–β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

scholarly journals Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 172 ◽  
Author(s):  
Braulio Vargas Möller-Hergt ◽  
Andreas Carlström ◽  
Tamara Suhm ◽  
Martin Ott
Keyword(s):  
Quality Control ◽  
Control System ◽  
Mitochondrial Protein ◽  
Quality Control System ◽  
Yeast Cells ◽  
Mitochondrial Quality Control ◽  
Protein Insertion ◽  
Mitochondrial Proteome ◽  
Proteotoxic Stress ◽  
Membrane Protein Insertion

The mitochondrial proteome contains proteins from two different genetic systems. Proteins are either synthesized in the cytosol and imported into the different compartments of the organelle or directly produced in the mitochondrial matrix. To ensure proteostasis, proteins are monitored by the mitochondrial quality control system, which will degrade non-native polypeptides. Defective mitochondrial membrane proteins are degraded by membrane-bound AAA-proteases. These proteases are regulated by factors promoting protein turnover or preventing their degradation. Here we determined genetic interactions between the mitoribosome receptors Mrx15 and Mba1 with the quality control system. We show that simultaneous absence of Mrx15 and the regulators of the i-AAA protease Mgr1 and Mgr3 provokes respiratory deficiency. Surprisingly, mutants lacking Mrx15 were more tolerant against proteotoxic stress. Furthermore, yeast cells became hypersensitive against proteotoxic stress upon deletion of MBA1. Contrary to Mrx15, Mba1 cooperates with the regulators of the m-AAA and i-AAA proteases. Taken together, these results suggest that membrane protein insertion and mitochondrial AAA-proteases are functionally coupled, possibly reflecting an early quality control step during mitochondrial protein synthesis.

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