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1.
Biochim Biophys Acta Mol Cell Res ; 1870(2): 119403, 2023 02.
Article in English | MEDLINE | ID: mdl-36427551

ABSTRACT

The membrane insertase YidC, is an essential bacterial component and functions in the folding and insertion of many membrane proteins during their biogenesis. It is a multispanning protein in the inner (cytoplasmic) membrane of Escherichia coli that binds its substrates in the "greasy slide" through hydrophobic interaction. The hydrophilic part of the substrate transiently localizes in the groove of YidC before it is translocated into the periplasm. The groove, which is flanked by the greasy slide, is within the center of the membrane, and provides a promising target for inhibitors that would block the insertase function of YidC. In addition, since the greasy slide is available for the binding of various substrates, it could also provide a binding site for inhibitory molecules. In this review we discuss in detail the structure and the mechanism of how YidC interacts not only with its substrates, but also with its partner proteins, the SecYEG translocase and the SRP signal recognition particle. Insight into the substrate binding to the YidC catalytic groove is presented. We wind up the review with the idea that the hydrophilic groove would be a potential site for drug binding and the feasibility of YidC-targeted drug development.


Subject(s)
Escherichia coli Proteins , Membrane Transport Proteins , Membrane Transport Proteins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Proteins/metabolism , Cell Membrane/metabolism
2.
Front Physiol ; 13: 933153, 2022.
Article in English | MEDLINE | ID: mdl-35957980

ABSTRACT

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

3.
J Biol Chem ; 298(7): 102107, 2022 07.
Article in English | MEDLINE | ID: mdl-35671825

ABSTRACT

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.


Subject(s)
Protein Folding , Protein Transport , Bacteria/metabolism , Bacterial Proteins/metabolism , Eukaryota/metabolism , Membrane Transport Proteins/metabolism , Peroxisomes/metabolism , Protein Sorting Signals , Twin-Arginine-Translocation System/metabolism
4.
J Biol Chem ; 298(3): 101690, 2022 03.
Article in English | MEDLINE | ID: mdl-35148995

ABSTRACT

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.


Subject(s)
Escherichia coli Proteins , Membrane Transport Proteins , Bacillus subtilis/enzymology , Cell Membrane/metabolism , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Structure-Activity Relationship
5.
Sci Rep ; 11(1): 3940, 2021 02 16.
Article in English | MEDLINE | ID: mdl-33594158

ABSTRACT

The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.


Subject(s)
Escherichia coli Proteins/metabolism , Membrane Transport Proteins/metabolism , SEC Translocation Channels/metabolism , Escherichia coli , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Membrane Transport Proteins/genetics , Membrane Transport Proteins/isolation & purification , Proteolipids/metabolism , SEC Translocation Channels/isolation & purification
6.
Biochim Biophys Acta Biomembr ; 1863(2): 183502, 2021 02 01.
Article in English | MEDLINE | ID: mdl-33130098

ABSTRACT

The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.


Subject(s)
Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Membrane Transport Proteins/metabolism , Periplasm/metabolism , SEC Translocation Channels/metabolism , Animals , Cell Line , Cell Membrane/chemistry , Cell Membrane/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Mice , Periplasm/chemistry , Periplasm/genetics , Protein Structure, Secondary , SEC Translocation Channels/chemistry , SEC Translocation Channels/genetics
7.
J Mol Biol ; 432(2): 484-496, 2020 01 17.
Article in English | MEDLINE | ID: mdl-31669168

ABSTRACT

Proper membrane insertion is crucial for the structure and function of membrane proteins in all cells. The YidC insertase plays an essential role in this process, but the molecular mechanism of YidC-mediated insertion remains unknown. Here we track the stepwise movement of Pf3 coat through YidC by obtaining a series of translational arrested intermediates, and investigate them by thiol cross-linking. We show that Pf3 is inserted as a helical hairpin, i.e., the prospective transmembrane segment moves along the YidC greasy slide comprised of TM3 and TM5, whereas the N-terminal tail transiently folds back into the hydrophilic groove of YidC located in the inner leaflet of the membrane until it is translocated to the periplasm in a subsequent step involving the electrochemical membrane potential. In addition to providing virtual insights about how YidC inserts single-spanning membrane proteins, our study also demonstrates a valuable in vivo tracking method that can be applied to study more complicated substrates or other translocases.


Subject(s)
Escherichia coli Proteins/ultrastructure , Escherichia coli/ultrastructure , Membrane Proteins/ultrastructure , Membrane Transport Proteins/ultrastructure , Protein Biosynthesis , Cell Membrane/genetics , Cell Membrane/ultrastructure , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Hydrophobic and Hydrophilic Interactions , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Mutation/genetics , Periplasm/genetics , Protein Transport/genetics , Substrate Specificity
9.
Protein J ; 38(3): 236-248, 2019 06.
Article in English | MEDLINE | ID: mdl-31187382

ABSTRACT

The past several decades have witnessed tremendous growth in the protein targeting, transport and translocation field. Major advances were made during this time period. Now the molecular details of the targeting factors, receptors and the membrane channels that were envisioned in Blobel's Signal Hypothesis in the 1970s have been revealed by powerful structural methods. It is evident that there is a myriad of cytosolic and membrane associated systems that accurately sort and target newly synthesized proteins to their correct membrane translocases for membrane insertion or protein translocation. Here we will describe the common principles for protein transport in prokaryotes and eukaryotes.


Subject(s)
Molecular Chaperones/physiology , Protein Sorting Signals , Protein Translocation Systems , Proteins/metabolism , Escherichia coli/metabolism , Molecular Chaperones/chemistry , Protein Translocation Systems/chemistry , Protein Translocation Systems/physiology , Protein Transport , SEC Translocation Channels/chemistry , Yeasts/metabolism
10.
Microbiol Spectr ; 7(1)2019 01.
Article in English | MEDLINE | ID: mdl-30761982

ABSTRACT

YidC insertase plays a pivotal role in the membrane integration, folding, and assembly of a number of proteins, including energy-transducing respiratory complexes, both autonomously and in concert with the SecYEG channel in bacteria. The YidC family of proteins is widely conserved in all domains of life, with new members recently identified in the eukaryotic endoplasmic reticulum membrane. Bacterial and organellar members share the conserved 5-transmembrane core, which forms a unique hydrophilic cavity in the inner leaflet of the bilayer accessible from the cytoplasm and the lipid phase. In this chapter, we discuss the YidC family of proteins, focusing on its mechanism of substrate insertion independently and in association with the Sec translocon.


Subject(s)
Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Membrane Transport Proteins/metabolism , Bacillus subtilis/metabolism , Biological Transport/physiology , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/metabolism , SEC Translocation Channels/physiology
11.
J Mol Biol ; 431(5): 1025-1037, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30639187

ABSTRACT

Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion, and assembly of inner-membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In this study, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36-residue N-tail of 2Pf3-Lep translocates independent of YidC-Sec. This is the longest known substrate of this pathway. We confirmed this using a newly constructed YidC-Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC-Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted; however, an MBP-2Pf3-Lep fusion protein could be ranslocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC-Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in Escherichia coli.


Subject(s)
Escherichia coli Proteins/metabolism , Membrane Transport Proteins/metabolism , Protein Transport/physiology , SEC Translocation Channels/metabolism , Cell Membrane/metabolism , Escherichia coli/metabolism , Membrane Proteins/metabolism
12.
Sci Rep ; 8(1): 589, 2018 01 12.
Article in English | MEDLINE | ID: mdl-29330366

ABSTRACT

The membrane insertase YidC catalyzes the entrance of newly synthesized proteins into the lipid bilayer. As an integral membrane protein itself, YidC can be found as a monomer, a dimer or also as a member of the holotranslocase SecYEGDF-YajC-YidC. To investigate whether the dimeric YidC is functional and whether two copies cooperate to insert a single substrate, we constructed a fusion protein where two copies of YidC are connected by a short linker peptide. The 120 kDa protein is stable and functional as it supports the membrane insertion of the M13 procoat protein, the C-tailed protein SciP and the fusion protein Pf3-Lep. Mutations that inhibit either protomer do not inactivate the insertase and rather keep it functional. When both protomers are defective, the substrate proteins accumulate in the cytoplasm. This suggests that the dimeric YidC operates as two insertases. Consistent with this, we show that the dimeric YidC can bind two substrate proteins simultaneously, suggesting that YidC indeed functions as a monomer.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Mutation , Enzyme Stability , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Lipid Bilayers/metabolism , Membrane Transport Proteins/genetics , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
13.
Trends Biochem Sci ; 43(3): 151-153, 2018 03.
Article in English | MEDLINE | ID: mdl-29310909

ABSTRACT

Oxa1/Alb3/YidC family members promote the insertion of proteins into the mitochondrial inner membrane, the chloroplast thylakoid membrane, and the bacterial plasma membrane. Remarkably, two recent studies identify new Oxa1 homologs that reside in the endoplasmic reticulum (ER) and function in ER membrane protein biogenesis.


Subject(s)
Electron Transport Complex IV , Mitochondrial Proteins , Endoplasmic Reticulum , Escherichia coli Proteins , Membrane Transport Proteins , Mitochondrial Membranes , Nuclear Proteins
14.
Nat Rev Microbiol ; 16(2): 120, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29249813

ABSTRACT

This corrects the article DOI: 10.1038/nrmicro3595.

15.
Structure ; 25(9): 1403-1414.e3, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28844594

ABSTRACT

The YidC/Oxa1/Alb3 family of membrane proteins function to insert proteins into membranes in bacteria, mitochondria, and chloroplasts. Recent X-ray structures of YidC from Bacillus halodurans and Escherichia coli revealed a hydrophilic groove that is accessible from the lipid bilayer and the cytoplasm. Here, we explore the water accessibility within the conserved core region of the E. coli YidC using in vivo cysteine alkylation scanning and molecular dynamics (MD) simulations of YidC in POPE/POPG membranes. As expected from the structure, YidC possesses an aqueous membrane cavity localized to the membrane inner leaflet. Both the scanning data and the MD simulations show that the lipid-exposed transmembrane helices 3, 4, and 5 are short, leading to membrane thinning around YidC. Close examination of the MD data reveals previously unrecognized structural features that are likely important for protein stability and function.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Water/metabolism , Alkylation , Cell Membrane/metabolism , Crystallography, X-Ray , Cysteine/chemistry , Escherichia coli/chemistry , Models, Molecular , Molecular Dynamics Simulation , Protein Stability , Protein Structure, Secondary
16.
EcoSal Plus ; 7(2)2017 03.
Article in English | MEDLINE | ID: mdl-28276312

ABSTRACT

The insertion and assembly of proteins into the inner membrane of bacteria are crucial for many cellular processes, including cellular respiration, signal transduction, and ion and pH homeostasis. This process requires efficient membrane targeting and insertion of proteins into the lipid bilayer in their correct orientation and proper conformation. Playing center stage in these events are the targeting components, signal recognition particle (SRP) and the SRP receptor FtsY, as well as the insertion components, the Sec translocon and the YidC insertase. Here, we will discuss new insights provided from the recent high-resolution structures of these proteins. In addition, we will review the mechanism by which a variety of proteins with different topologies are inserted into the inner membrane of Gram-negative bacteria. Finally, we report on the energetics of this process and provide information on how membrane insertion occurs in Gram-positive bacteria and Archaea. It should be noted that most of what we know about membrane protein assembly in bacteria is based on studies conducted in Escherichia coli.


Subject(s)
Escherichia coli Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Archaea/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Gram-Positive Bacteria/metabolism , Membrane Proteins/chemistry , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Molecular Structure , Protein Transport , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Peptide/genetics , SEC Translocation Channels , Signal Recognition Particle/genetics , Signal Recognition Particle/metabolism
17.
Curr Biol ; 26(17): R811-3, 2016 09 12.
Article in English | MEDLINE | ID: mdl-27623265

ABSTRACT

A new study of the bacterial Sec translocase complex reports that ADP/ATP binding to SecA triggers multiple conformational changes in the SecYEG channel that may allow the passive directional movement of the polypeptide chain.


Subject(s)
Membrane Transport Proteins , SEC Translocation Channels , Adenosine Triphosphatases , Bacterial Proteins , Protein Transport
18.
Structure ; 23(9): 1559-1560, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26331454

ABSTRACT

In this issue of Structure, Borowska et al. (2015) report the crystal structure and provide experimental evidence on an archaeal membrane insertase, the DUF106 protein from Methanocaldococcus jannaschii, demonstrating that YidC/Oxa1/Alb3-like insertases exist in the archaeal plasma membrane.


Subject(s)
Cell Membrane/metabolism , Membrane Transport Proteins/chemistry , Methanocaldococcus/metabolism
19.
J Biol Chem ; 290(24): 14866-74, 2015 Jun 12.
Article in English | MEDLINE | ID: mdl-25947384

ABSTRACT

The YidC/Alb3/Oxa1 family functions in the insertion and folding of proteins in the bacterial cytoplasmic membrane, the chloroplast thylakoid membrane, and the mitochondrial inner membrane. All members share a conserved region composed of five transmembrane regions. These proteins mediate membrane insertion of an assorted group of proteins, ranging from respiratory subunits in the mitochondria and light-harvesting chlorophyll-binding proteins in chloroplasts to ATP synthase subunits in bacteria. This review discusses the YidC/Alb3/Oxa1 protein family as well as their function in membrane insertion and two new structures of the bacterial YidC, which suggest a mechanism for membrane insertion by this family of insertases.


Subject(s)
Membrane Proteins/metabolism , beta-Fructofuranosidase/metabolism , Substrate Specificity
20.
J Mol Biol ; 427(5): 1023-37, 2015 Mar 13.
Article in English | MEDLINE | ID: mdl-24846669

ABSTRACT

The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell's targeting mechanisms can readily identify it as it emerges from the ribosome. However, the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ's novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immunotags before the TM domain to assess membrane insertion using proteinase K digestion. We confirmed the N(in)-C(out) (type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the TM proton gradient was abolished with Carbonyl cyanide m-chlorophenyl hydrazone. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the TM proton motive force.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cytoskeletal Proteins/metabolism , Escherichia coli Proteins/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Escherichia coli/metabolism , Protein Transport/physiology , Ribosomes/metabolism , SEC Translocation Channels , SecA Proteins , Signal Recognition Particle/metabolism
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