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 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 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.

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 Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes inStreptomyces coelicolor

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Meng Liu ◽  
Peipei Zhang ◽  
Yanping Zhu ◽  
Ting Lu ◽  
Yemin Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Consensus Sequence ◽  
Aerial Mycelium ◽  
Dnase I ◽  
Inverted Repeats ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Two Component

ABSTRACTAs with most annotated two-component systems (TCSs) ofStreptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, formorphogenesis andactinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using anS. coelicolorstrain expressing MacR-Flag fusion protein, identifiedin vivotargets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assaysin vitroand by ChIP-quantitative PCRin vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes,sco6728,sco4924, andsco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS fromS. avermitilisandS. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism inStreptomyces.IMPORTANCETCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in theStreptomycesmodel strainS. coelicolorare unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that otherStreptomycesspecies have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems inStreptomyces.

scholarly journals A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

2009 ◽  
Vol 28 (22) ◽  
pp. 3461-3475 ◽  
Author(s):  
Shinobu Chiba ◽  
Anne Lamsa ◽  
Kit Pogliano
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Protein ◽  
Protein Biogenesis ◽  
Nascent Chain

scholarly journals Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

2017 ◽  
Vol 199 (9) ◽  
Author(s):  
Yunxue Guo ◽  
Xiaoxiao Liu ◽  
Baiyuan Li ◽  
Jianyun Yao ◽  
Thomas K. Wood ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Transmembrane Domain ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

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 Mutations of the YidC Insertase alleviate stress from σM-dependent membrane protein overproduction in Bacillus subtilis

2019 ◽  
Author(s):  
Heng Zhao ◽  
Ankita J. Sachla ◽  
John D. Helmann
Keyword(s):  
Oxidative Stress ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Substrate Binding ◽  
Single Amino Acid ◽  
Cell Wall Synthesis ◽  
Intrinsic Resistance ◽  
Σ Factor

AbstractIn Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidCQ140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.Author SummaryBacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the σM regulon, resulting from loss of the YhdL anti-σM protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high σM activity. Further analyses reveal that induction of the σM regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the σM regulon.

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