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 SRP19 Is a Dispensable Component of the Signal Recognition Particle in Archaea

2006 ◽  
Vol 189 (1) ◽  
pp. 276-279 ◽  
Author(s):  
Sophie Yurist ◽  
Idit Dahan ◽  
Jerry Eichler
Keyword(s):  
Cell Growth ◽  
Membrane Protein ◽  
Protein Secretion ◽  
Signal Recognition Particle ◽  
Haloferax Volcanii ◽  
Signal Recognition ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Srp Rna

ABSTRACT In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.

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 F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

2004 ◽  
Vol 165 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Martin van der Laan ◽  
Philipp Bechtluft ◽  
Stef Kol ◽  
Nico Nouwen ◽  
Arnold J.M. Driessen
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Atp Synthase ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Membrane Insertion ◽  
Subunit C ◽  
Protein Insertion ◽  
F1f0 Atp Synthase

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro–synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.

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 ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in Drosophila photoreceptors

2019 ◽  
Vol 30 (23) ◽  
pp. 2890-2900 ◽  
Author(s):  
Naoki Hiramatsu ◽  
Tatsuya Tago ◽  
Takunori Satoh ◽  
Akiko K. Satoh
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complex ◽  
Model System ◽  
Membrane Insertion ◽  
Transmembrane Helices ◽  
Membrane Protein Complex ◽  
C Terminus ◽  
Wide Range ◽  
Er Membrane

Most membrane proteins are synthesized on and inserted into the membrane of the endoplasmic reticulum (ER), in eukaryote. The widely conserved ER membrane protein complex (EMC) facilitates the biogenesis of a wide range of membrane proteins. In this study, we investigated the EMC function using Drosophila photoreceptor as a model system. We found that the EMC was necessary only for the biogenesis of a subset of multipass membrane proteins such as rhodopsin (Rh1), TRP, TRPL, Csat, Cni, SERCA, and Na+K+ATPase α, but not for that of secretory or single-pass membrane proteins. Additionally, in EMC-deficient cells, Rh1 was translated to its C terminus but degraded independently from ER-associated degradation. Thus, EMC exerted its effect after translation but before or during the membrane integration of transmembrane domains (TMDs). Finally, we found that EMC was not required for the stable expression of the first three TMDs of Rh1 but was required for that of the fourth and fifth TMDs. Our results suggested that EMC is required for the ER membrane insertion of succeeding TMDs of multipass membrane proteins.

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.

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