| 1. |
- Ariöz, Candan, 1983-, et al.
(författare)
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Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:33, s. 5533-5544
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Tidskriftsartikel (refereegranskat)abstract
- Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
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| 2. |
- Ariöz, Candan, 1983-, et al.
(författare)
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Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes :
- 2014
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1838:7, s. 1862-1870
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Tidskriftsartikel (refereegranskat)abstract
- The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.
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| 3. |
- Götzke, Hansjörg, et al.
(författare)
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Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
- 2015
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 14:1, s. 216-226
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Tidskriftsartikel (refereegranskat)abstract
- How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.
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| 4. |
- Götzke, Hansjörg, 1984-
(författare)
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Protein trafficking in the cell envelope of Escherichia coli : Identification and characterisation of a novel chaperone
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell envelope of Gram-negative bacteria, like Escherichia coli, is composed of a cytoplasmic membrane, a periplasmic space containing a peptidoglycan layer and an outer membrane. About 30 % of all proteins are localised in the cell envelope. These proteins have to be inserted into or translocated across the inner membrane by the SecYEG translocon. They are then chaperoned to their final destination by a network of chaperones. The broad aim of this work was to provide a better understanding of protein trafficking through the bacterial cell envelope. We have identified a novel membrane protein complex consisting of the periplasmic chaperone PpiD and the uncharacterised protein YfgM. Both are anchored in the inner membrane and have periplasmic domains. By co-immunoprecipitations and two-dimensional gel electrophoresis it could be demonstrated that YfgM and PpiD form a supercomplex with the SecYEG translocon. Furthermore, a chemical-genetic approach showed that YfgM is part of the periplasmic chaperone network that is essential for envelope protein biogenesis. Moreover, it could be shown that YfgM is required for the stability of the periplasmic chaperone HdeB. Finally, evidence that YfgM might also be involved in the lateral insertion of transmembrane domains was provided. In summary, this thesis details the identification and characterisation of a novel ancillary subunit of the SecYEG translocon that is involved in the periplasmic chaperone network in the cell envelope of Escherichia coli.
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| 5. |
- Götzke, Hansjörg, et al.
(författare)
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The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.
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| 6. |
- Götzke, Hansjörg, et al.
(författare)
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YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
- 2014
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:27, s. 19089-19097
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Tidskriftsartikel (refereegranskat)abstract
- Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.
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| 7. |
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| 8. |
- Maddalo, Gianluca, et al.
(författare)
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Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
- 2011
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1848-1859
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Tidskriftsartikel (refereegranskat)abstract
- The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
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| 9. |
- Muheim, Claudio, et al.
(författare)
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Increasing the permeability of Escherichia coli using MAC13243
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.
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| 10. |
- Muheim, Claudio, et al.
(författare)
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Increasing the permeability of Escherichia coli using MAC13243
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.
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| 11. |
- Norell, Derrick, et al.
(författare)
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Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 5396-
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Tidskriftsartikel (refereegranskat)abstract
- Gram-negative bacteria use the type-V secretion pathway to expose proteins at their cell surface, many of which have virulence functions. Translocation of those proteins across the outer membrane occurs either by means of dedicated translocator proteins (two-partner secretion) or covalently fused translocator domains (autotransporters). Translocator proteins and translocator domains are beta-barrels requiring the beta-barrel assembly machinery (BAM) for membrane integration. However, the molecular details of their passage across the envelope and insertion into the outer membrane remain enigmatic, owing in part to the fact that in vitro systems are not available. Here we describe a versatile in vitro reconstitution system that faithfully reproduces both branches of the type-V secretion pathway and the assembly of beta-barrel outer membrane proteins. This system will allow an in-depth analysis of protein secretion across and integration into outer membranes.
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