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Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2014. , 73 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-106277ISBN: 978-91-7447-944-7 (print)OAI: oai:DiVA.org:su-106277DiVA: diva2:735676
Public defence
2014-09-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2014-09-03 Created: 2014-07-30 Last updated: 2017-10-30Bibliographically approved
List of papers
1. Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
Open this publication in new window or tab >>Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1848-1859 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Escherichia coli, cell envelope, proteome, membrane protein, protein complex, BN-PAGE, PpiD, YfgM
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-56162 (URN)10.1021/pr101105c (DOI)000288924000038 ()21210718 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
2. YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
Open this publication in new window or tab >>YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 27, 19089-19097 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106016 (URN)10.1074/jbc.M113.541672 (DOI)000339062900039 ()
Available from: 2014-07-18 Created: 2014-07-18 Last updated: 2017-12-05Bibliographically approved
3. Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
Open this publication in new window or tab >>Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
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2015 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 1, 216-226 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-113555 (URN)10.1074/mcp.M114.043216 (DOI)000347155500017 ()
Note

AuthorCount:6;

Available from: 2015-02-06 Created: 2015-02-04 Last updated: 2017-12-04Bibliographically approved
4. YfgM plays a role in protein insertion through the SecYEG translocon
Open this publication in new window or tab >>YfgM plays a role in protein insertion through the SecYEG translocon
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106274 (URN)
Available from: 2014-07-29 Created: 2014-07-29 Last updated: 2014-08-01

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