scholarly journals EMC is required for biogenesis and membrane insertion of Xport-A, an essential chaperone of rhodopsin-1 and the TRP channel

2021 ◽  
Author(s):  
Catarina J. Gaspar ◽  
Lígia C. Vieira ◽  
John C. Christianson ◽  
David Jakubec ◽  
Kvido Strisovsky ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Bioinformatic Analysis ◽  
Trp Channel ◽  
Membrane Insertion ◽  
Loss Of Function ◽  
Membrane Complex ◽  
Protein Biogenesis ◽  
Eukaryotic Membrane Protein ◽  
Ta Proteins

SUMMARYInsertion of hydrophobic transmembrane domains (TMDs) into the endoplasmic reticulum (ER) lipid bilayer is an essential step during eukaryotic membrane protein biogenesis. The ER membrane complex (EMC) functions as an insertase for TMDs of low hydrophobicity and is required for the biogenesis of a subset of tail-anchored (TA) and polytopic membrane proteins, including rhodopsin-1 (Rh1) and the TRP channel. To better understand the physiological implications of membrane protein biogenesis dependent on the EMC, we performed a bioinformatic analysis to predict TA proteins present in the Drosophila proteome. From 254 predicted TA proteins, subsequent genetic screening in Drosophila larval eye discs led to the identification of 2 proteins that require EMC for their biogenesis: farinelli (fan) and Xport-A. Fan is required for sperm individualization and male fertility in Drosophila and we now show that EMC is also required for these important biological processes. Interestingly, Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC loss of function mutations is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and increasing the hydrophobicity of Xport-A TMD rendered its membrane insertion to become EMC-independent. Moreover, these EMC-independent Xport-A mutants rescued Rh1 and TRP biogenesis in EMC mutants. Our data establish that EMC can impact the biogenesis of polytopic membrane proteins indirectly, by controlling the biogenesis and membrane insertion of an essential protein co-factor.

scholarly journals MifM Monitors Total YidC Activities of Bacillus subtilis, Including That of YidC2, the Target of Regulation

2014 ◽  
Vol 197 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Shinobu Chiba ◽  
Koreaki Ito
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Membrane Insertion ◽  
Translation Arrest ◽  
Content Type ◽  
Ribosome Stalling ◽  
Protein Biogenesis ◽  
Independent Mechanism ◽  
Charged Residues

The YidC/Oxa1/Alb3 family proteins are involved in membrane protein biogenesis in bacteria, mitochondria, and chloroplasts. Recent studies show that YidC uses a channel-independent mechanism to insert a class of membrane proteins into the membrane.Bacillus subtilishas two YidC homologs, SpoIIIJ (YidC1) and YidC2 (YqjG); the former is expressed constitutively, while the latter is induced when the SpoIIIJ activity is compromised. MifM is a substrate of SpoIIIJ, and its failure in membrane insertion is accompanied by stable ribosome stalling on themifM-yidC2mRNA, which ultimately facilitatesyidC2translation. While mutational inactivation of SpoIIIJ has been known to induceyidC2expression, here, we show that the level of this induction is lower than that observed when the membrane insertion signal of MifM is defective. Moreover, this partial induction of YidC2 translation is lowered further when YidC2 is overexpressed intrans. These results suggest that YidC2 is able to insert MifM into the membrane and to release its translation arrest. Thus, under SpoIIIJ-deficient conditions, YidC2 expression is subject to MifM-mediated autogenous feedback repression. Our results show that YidC2 uses a mechanism that is virtually identical to that used by SpoIIIJ; Arg75 of YidC2 in its intramembrane yet hydrophilic cavity is functionally indispensable and requires negatively charged residues of MifM as an insertion substrate. From these results, we conclude that MifM monitors the total activities of the SpoIIIJ and the YidC2 pathways to control the synthesis of YidC2 and to maintain the cellular capability of the YidC mode of membrane protein biogenesis.

scholarly journals YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

2004 ◽  
Vol 166 (6) ◽  
pp. 769-774 ◽  
Author(s):  
Ross E. Dalbey ◽  
Andreas Kuhn
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Family Members ◽  
Membrane Insertion ◽  
Conducting Channel ◽  
Energy Devices ◽  
Domains Of Life ◽  
Membrane Protein Complexes ◽  
Sensor Proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein–conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.

scholarly journals An ER translocon for multi-pass membrane protein biogenesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Philip T McGilvray ◽  
S Andrei Anghel ◽  
Arunkumar Sundaram ◽  
Frank Zhong ◽  
Michael J Trnka ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Glutamate Transporter ◽  
Cell Physiology ◽  
Transmembrane Segments ◽  
Cryo Electron Microscopy ◽  
Protein Biogenesis ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Molecular Framework

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

scholarly journals Evidence for Membrane Complex Assembly in Nanoelectrospray Generated Lipid Bilayers

2019 ◽  
Author(s):  
Matthias Wilm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Layered Structure ◽  
Protein Complexes ◽  
Complex Assembly ◽  
Membrane Complex ◽  
Detergent Extraction ◽  
Membrane Protein Complexes

1.AbstractNanoelectrospray can be used to generate a layered structure consisting of bipolar lipids, detergent-solubilized membrane proteins, and glycerol that self-assembles upon detergent extraction into one extended layer of a protein containing membrane. This manuscript presents the first evidence that this method might allow membrane protein complexes to assemble in this process.

scholarly journals Unbalanced Charge Distribution as a Determinant for Dependence of a Subset of Escherichia coli Membrane Proteins on the Membrane Insertase YidC

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Andrew N. Gray ◽  
Josephine M. Henderson-Frost ◽  
Dana Boyd ◽  
Shirin Shirafi ◽  
Hironori Niki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Charge Distribution ◽  
Biological Membranes ◽  
Membrane Insertion ◽  
Content Type ◽  
A Genome ◽  
Cell Processes ◽  
A Charge

ABSTRACTMembrane proteins are involved in numerous essential cell processes, including transport, gene regulation, motility, and metabolism. To function properly, they must be inserted into the membrane and folded correctly. YidC, an essential protein inEscherichia coliwith homologues in other bacteria,Archaea, mitochondria, and chloroplasts, functions by incompletely understood mechanisms in the insertion and folding of certain membrane proteins. Using a genome-scale approach, we identified 69E. colimembrane proteins that, in the absence of YidC, exhibited aberrant localization by microscopy. Further examination of a subset revealed biochemical defects in membrane insertion in the absence of YidC, indicating their dependence on YidC for proper membrane insertion or folding. Membrane proteins possessing an unfavorable distribution of positively charged residues were significantly more likely to depend on YidC for membrane insertion. Correcting the charge distribution of a charge-unbalanced YidC-dependent membrane protein abrogated its requirement for YidC, while perturbing the charge distribution of a charge-balanced YidC-independent membrane protein rendered it YidC dependent, demonstrating that charge distribution can be a necessary and sufficient determinant of YidC dependence. These findings provide insights into a mechanism by which YidC promotes proper membrane protein biogenesis and suggest a critical function of YidC in all organisms and organelles that express it.IMPORTANCEBiological membranes are fundamental components of cells, providing barriers that enclose the cell and separate compartments. Proteins inserted into biological membranes serve critical functions in molecular transport, molecular partitioning, and other essential cell processes. The mechanisms involved in the insertion of proteins into membranes, however, are incompletely understood. The YidC protein is critical for the insertion of a subset of proteins into membranes across an evolutionarily wide group of organisms. Here we identify a large group of proteins that depend on YidC for membrane insertion inEscherichia coli, and we identify unfavorable distribution of charge as an important determinant of YidC dependence for proper membrane insertion.

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 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 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 Bacillus subtilis SpoIIIJ and YqjG Function in Membrane Protein Biogenesis

2009 ◽  
Vol 191 (21) ◽  
pp. 6749-6757 ◽  
Author(s):  
Manfred J. Saller ◽  
Fabrizia Fusetti ◽  
Arnold J. M. Driessen
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Protein ◽  
Atp Synthase ◽  
Protein Secretion ◽  
Model Organism ◽  
Membrane Vesicles ◽  
Membrane Insertion ◽  
E Coli ◽  
Protein Biogenesis ◽  
Domains Of Life

ABSTRACT In all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis. Bacillus subtilis, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either spoIIIJ or yqjG functionally compensates for the defects in membrane insertion due to YidC depletion in Escherichia coli. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome o oxidase and F1Fo ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F1Fo ATP synthase subunit c from both E. coli and B. subtilis into inner membrane vesicles of E. coli. When isolated from B. subtilis cells, SpoIIIJ and YqjG were found to be associated with the entire F1Fo ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the Bacillus Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.

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