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Membrane Protein Biogenesis in Escherichia coli: A proteomics approach
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Jan-Willem de Gier)
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 [en]
SRP, YidC, SecE, Membrane protein biogenesis, protein targeting, 2D BN/SDS-PAGE
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-36851ISBN: 978-91-7447-008-6 (print)OAI: oai:DiVA.org:su-36851DiVA: diva2:290606
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
List of papers
1. Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli
Open this publication in new window or tab >>Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli
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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
Keyword
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:nbn:se:su:diva-16865 (URN)10.1128/JB.01631-07 (DOI)000255622500011 ()18296516 (PubMedID)
Available from: 2008-12-29 Created: 2008-12-29 Last updated: 2017-12-13Bibliographically approved
2. Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
Open this publication in new window or tab >>Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
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2008 (English)In: Methods, ISSN 1095-9130, Vol. 46, no 2, 48-53 p.Article in journal (Refereed) Published
Abstract [en]

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.

Keyword
rane protein, Protein complex, Two-dimensional blue native SDS–PAGE, Comparative proteomics, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-17270 (URN)10.1016/j.ymeth.2008.06.017 (DOI)000261367600002 ()18674622 (PubMedID)
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2011-06-15Bibliographically approved

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