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

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00057-17 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 11

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.

Download Full-text

Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

mBio ◽

10.1128/mbio.01580-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 1

Author(s):

Markus Peschke ◽

Mélanie Le Goff ◽

Gregory M. Koningstein ◽

Norbert O. Vischer ◽

Abbi Abdel-Rehim ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Transmembrane Domain ◽

Signal Recognition Particle ◽

Membrane Insertion ◽

Type Ii ◽

Content Type ◽

E Coli ◽

C Terminus

ABSTRACT Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented. IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus “regular” membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

Download Full-text

Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed

mBio ◽

10.1128/mbio.00020-10 ◽

2010 ◽

Vol 1 (2) ◽

Cited By ~ 11

Author(s):

Ido Yosef ◽

Elena S. Bochkareva ◽

Eitan Bibi

Keyword(s):

Escherichia Coli ◽

Quality Control ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Translation ◽

Mrna Levels ◽

Content Type ◽

E Coli ◽

Quality Control Process

ABSTRACT The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.02074-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 99-107 ◽

Cited By ~ 16

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.

Download Full-text

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501836112 ◽

2015 ◽

Vol 112 (31) ◽

pp. 9632-9637 ◽

Cited By ~ 15

Author(s):

James A. Stapleton ◽

Timothy A. Whitehead ◽

Vikas Nanda

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Membrane Proteins ◽

Cell Membrane ◽

Membrane Protein ◽

Computational Design ◽

Residue Level ◽

Membrane Insertion ◽

Amino Acid Residues ◽

Design Rules

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface ofEscherichia coliOmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.02178-18 ◽

2018 ◽

Vol 85 (4) ◽

Cited By ~ 5

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.

Download Full-text

Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

Microbiology Resource Announcements ◽

10.1128/mra.00804-19 ◽

2019 ◽

Vol 8 (32) ◽

Author(s):

Yen-Te Liao ◽

Yujie Zhang ◽

Alexandra Salvador ◽

Vivian C. H. Wu

Keyword(s):

Escherichia Coli ◽

Surface Water ◽

Genome Size ◽

Shiga Toxin ◽

Open Reading Frames ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

A Genome ◽

Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

Download Full-text

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

Journal of Bacteriology ◽

10.1128/jb.01493-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 220-230 ◽

Cited By ~ 11

Author(s):

Jun Liu ◽

David B. Hicks ◽

Terry A. Krulwich

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Atpase Activity ◽

Atp Synthase ◽

Membrane Association ◽

Functional Specialization ◽

Chromosomal Deletion ◽

Content Type ◽

Oligomer Formation ◽

Alkaliphilic Bacillus

ABSTRACTAtpI, a membrane protein encoded by many bacterialatpoperons, is reported to be necessary forc-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream ofatpIthat has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilicBacillus pseudofirmusOF4. A strain with a chromosomal deletion ofatpIgrew nonfermentatively, and its purified ATP synthase had ac-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion ofatpI, but notatpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F1domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. BothspoIIIJandyqjG, but notatpIoratpZ, complemented a YidC-depletedEscherichia colistrain. Consistent with such overlapping functions, single deletions ofspoIIIJoryqjGin the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression ofyqjGwas elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJstrains, but it was lower than constitutivespoIIIJexpression. Deletion ofatpZcaused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.

Download Full-text

Glycolipozyme membrane protein integrase (MPIase): recent data

BioMolecular Concepts ◽

10.1515/bmc-2014-0030 ◽

2014 ◽

Vol 5 (5) ◽

pp. 429-438 ◽

Cited By ~ 10

Author(s):

Ken-ichi Nishiyama ◽

Keiko Shimamoto

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Membrane Protein ◽

Structure Determination ◽

Molecular Chaperone ◽

Molecular Mechanisms ◽

Cytoplasmic Membrane ◽

Amino Sugars ◽

Glycan Chain ◽

Preprotein Translocation

AbstractA novel factor for membrane protein integration, from the cytoplasmic membrane of Escherichia coli, named MPIase (membrane protein integrase), has recently been identified and characterized. MPIase was revealed to be essential for the membrane integration of a subset of membrane proteins, despite that such integration reactions have been, thus far, thought to occur spontaneously. The structure determination study revealed that MPIase is a novel glycolipid comprising a glycan chain with three N-acetylated amino sugars connected to diacylglycerol through a pyrophosphate linker. As MPIase catalyzes membrane protein integration, we propose that MPIase is a glycolipozyme on the basis of its enzyme-like function. The glycan chain exhibits a molecular chaperone-like function by directly interacting with substrate membrane proteins. Moreover, MPIase also affects the dimer structure of SecYEG, a translocon, thereby significantly stimulating preprotein translocation. The molecular mechanisms of MPIase functions will be outlined.

Download Full-text