scholarly journals A bacterial secretosome for regulated envelope biogenesis and quality control

2022 ◽  
Author(s):  
Daniel William Watkins ◽  
Ian Collinson
Keyword(s):  
Quality Control ◽  
Outer Membrane ◽  
Protein Delivery ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Inner Membrane ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Sec Translocon ◽  
Complex Forms

As the first line of defence against antibiotics, the Gram-negative bacterial envelope and its biogenesis are of considerable interest to the microbiological and biomedical communities. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed to the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core heterotrimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this secretosome: a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these and other factors act to ensure efficient whole envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. This organisation would be essential for cell wall biogenesis and remodelling and thus its perturbation would be a good strategy for the development of anti-microbials.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Effects of SecE Depletion on the Inner and Outer Membrane Proteomes of Escherichia coli

2008 ◽  
Vol 190 (10) ◽  
pp. 3505-3525 ◽  
Author(s):  
Louise Baars ◽  
Samuel Wagner ◽  
David Wickström ◽  
Mirjam Klepsch ◽  
A. Jimmy Ytterberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Identification ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Specific Model ◽  
Secretory Proteins ◽  
Inner Membrane ◽  
Sec Translocon

ABSTRACT The Sec translocon is a protein-conducting channel that allows polypeptides to be transferred across or integrated into a membrane. Although protein translocation and insertion in Escherichia coli have been studied using only a small set of specific model substrates, it is generally assumed that most secretory proteins and inner membrane proteins use the Sec translocon. Therefore, we have studied the role of the Sec translocon using subproteome analysis of cells depleted of the essential translocon component SecE. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and extensive immunoblotting. The analysis showed that upon SecE depletion (i) secretory proteins aggregated in the cytoplasm and the cytoplasmic σ32 stress response was induced, (ii) the accumulation of outer membrane proteins was reduced, with the exception of OmpA, Pal, and FadL, and (iii) the accumulation of a surprisingly large number of inner membrane proteins appeared to be unaffected or increased. These proteins lacked large translocated domains and/or consisted of only one or two transmembrane segments. Our study suggests that several secretory and inner membrane proteins can use Sec translocon-independent pathways or have superior access to the remaining Sec translocons present in SecE-depleted cells.

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.

scholarly journals Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

2012 ◽  
Vol 23 (20) ◽  
pp. 3948-3956 ◽  
Author(s):  
Maria Bohnert ◽  
Lena-Sophie Wenz ◽  
Ralf M. Zerbes ◽  
Susanne E. Horvath ◽  
David A. Stroud ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Early Stage ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Large Core ◽  
Large Protein ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Protein Biogenesis

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.

Membrane protein import in yeast mitochondria

2000 ◽  
Vol 28 (4) ◽  
pp. 495-499 ◽  
Author(s):  
K. Tokatlidis ◽  
S. Vial ◽  
P. Luciano ◽  
M. Vergnolle ◽  
S. Clémence
Keyword(s):  
Membrane Protein ◽  
Molecular Mechanism ◽  
Protein Import ◽  
Mitochondrial Matrix ◽  
Yeast Mitochondria ◽  
Inner Membrane ◽  
Protein Insertion ◽  
The Past ◽  
Membrane Protein Insertion ◽  
Membrane Bound

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn2+-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous inter-membrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

Periplasmic quality control in biogenesis of outer membrane proteins

2015 ◽  
Vol 43 (2) ◽  
pp. 133-138 ◽  
Author(s):  
Zhi Xin Lyu ◽  
Xin Sheng Zhao
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Diverse Range ◽  
Multifunctional Protein ◽  
Proposed Model ◽  
Periplasmic Proteins ◽  
Basic Functions ◽  
Biochemical Analyses

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

Sign in / Sign up

Export Citation Format

Share Document