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Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Show others and affiliations
2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 10, 3505-3525 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2008. Vol. 190, no 10, 3505-3525 p.
Keyword [en]
Bacterial Outer Membrane Proteins/metabolism, Cell Membrane/*metabolism, Escherichia coli/*genetics/metabolism, Escherichia coli Proteins/chemistry/*metabolism, Mass Spectrometry, Membrane Transport Proteins/chemistry/metabolism/*physiology, Proteome/*metabolism, Proteomics/*methods
Identifiers
URN: urn:nbn:se:su:diva-16865DOI: 10.1128/JB.01631-07ISI: 000255622500011PubMedID: 18296516OAI: oai:DiVA.org:su-16865DiVA: diva2:183385
Available from: 2008-12-29 Created: 2008-12-29 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Protein targeting, translocation and insertion in Escherichia coli: Proteomic analysis of substrate-pathway relationships
Open this publication in new window or tab >>Protein targeting, translocation and insertion in Escherichia coli: Proteomic analysis of substrate-pathway relationships
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Approximately 10% of the open reading frames in the genome of the Gram-negative bacterium E. coli encodes secretory proteins, and 20% encodes integral inner membrane proteins (IMPs). These proteins are sorted to their correct cellular compartments (the periplasm and the outer and inner membranes) by specialized targeting and translocation/insertion systems. So far, a very limited set of model proteins have been used to study proteins sorting requirements in E. coli. The main objective of all the papers presented in this thesis was to determine the targeting and translocation/insertion requirements of more E. coli proteins. In papers I and II, this was done using focused approaches. Selected model proteins (lipoproteins and putative outer membrane proteins) were expressed from plasmids and their targeting and translocation were analysed in vitro by crosslinking experiments and/or in vivo by pulse-chase analysis in different E. coli mutant strains. In papers III a comparative sub-proteome analysis was carried out to define the role of the cytoplasmic chaperone SecB in protein targeting. In paper IV, a similar approach was used to study how protein translocation and insertion is affected upon depletion of the essential Sec-translocon component SecE. The ‘global’ approach used in paper III and IV allowed us to study protein targeting and translocation/insertion requirements on a proteome level. This led to the identification of several novel SecB substrates and a large number of potential Sec-translocon independent IMPs.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2007. 132 p.
Keyword
protein biogenesis, targeting, translocation, insertion, SecB, SecE, E. coli
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-6990 (URN)978-91-7155-481-9 (ISBN)
Public defence
2007-09-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2007-08-13 Created: 2007-08-13 Last updated: 2009-05-12Bibliographically approved
2. From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli
Open this publication in new window or tab >>From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In both pro- and eukaryotes 20-30% of all genes encode alpha-helical transmembrane domain proteins, which act in various and often essential capacities. Notably, membrane proteins play key roles in disease and they constitute more than half of all known drug targets.

The natural abundance of membrane proteins is in general too low to conveniently isolate sufficient material for functional and structural studies. Therefore, most membrane proteins have to be obtained through overexpression. Escherichia coli is one of the most successful hosts for overexpression of recombinant proteins. While the production of soluble proteins is comparably straightforward, overexpression of membrane proteins remains a challenging task. The yield of membrane localized recombinant membrane protein is usually low and inclusion body formation is a serious problem. Furthermore, membrane protein overexpression is often toxic to the host cell. Although several reasons can be postulated, the basis of these difficulties is not completely understood, preventing the design of rational strategies to improve membrane protein overexpression yields.

The objective of my Ph.D. studies has been to improve membrane protein overexpression in E. coli by a) understanding membrane protein overexpression from the perspective of membrane protein biogenesis, b) systematically investigating the physiological response to overexpression of membrane proteins and c) engineering strains that are optimized for membrane protein overexpression based on insights resulting from these studies.

By working toward these objectives, I was able to identify and alleviate one of the major bottlenecks of membrane protein overexpression in E. coli: saturation of the Sec-translocon could be overcome by harmonizing translation and membrane insertion of the recombinant membrane protein. This minimized the toxic effects of overexpression and thus resulted in increased membrane protein-producing biomass.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 91 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-7513 (URN)978-91-7155-594-6 (ISBN)
Public defence
2008-05-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2010-01-13Bibliographically approved
3. Membrane Protein Biogenesis in Escherichia coli: A proteomics approach
Open this publication in new window or tab >>Membrane Protein Biogenesis in Escherichia coli: A proteomics approach
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the bacterium Escherichia coli all proteins are synthesized in the cytoplasm. However, at least 25% of them then need to be exported to another cellular compartment where they exert their function. E. coli has several different targeting pathways to ensure the correct localization of its proteins. When a nascent chain emerges at the ribosomal tunnel exit the signal recognition particle (SRP) can bind to it. The ribosomal nascent chain -SRP- complex is then targeted either to the Sec-translocon or the YidC insertase at the cytoplasmic membrane. Integral cytoplasmic membrane proteins are inserted into the lipid bilayer whilst periplasmic and outer membrane proteins are translocated across the membrane. In order to be fully functional, proteins need to be both correctly localized and folded. In many cases, they also assemble into complexes with other proteins or co-factors. In my thesis I will present an improved protocol for two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) that makes it very suitable to study the biogenesis of integral cytoplasmic membrane proteins and especially the complexes in the cytoplasmic membrane. Using this and other methods I have studied the biogenesis of integral cytoplasmic membrane proteins and other exported proteins. This has been done on a global scale in mutant strains where the SRP-targeting pathway, the Sec-translocon and the integral cytoplasmic membrane chaperone/insertase YidC have been compromised.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 99 p.
Keyword
SRP, YidC, SecE, Membrane protein biogenesis, protein targeting, 2D BN/SDS-PAGE
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-36851 (URN)978-91-7447-008-6 (ISBN)
Public defence
2010-02-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2010-02-04 Created: 2010-01-27 Last updated: 2010-01-28Bibliographically approved

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