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Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
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2007 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, 791-804 p.Article in journal (Refereed) Published
Abstract [en]

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI-TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.

Place, publisher, year, edition, pages
2007. Vol. 274, no 3, 791-804 p.
Keyword [en]
cyanobacteria, integral proteins, plasma membrane, proteome, Synechocystis 6803
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-56160DOI: 10.1111/j.1742-4658.2006.05624.xOAI: oai:DiVA.org:su-56160DiVA: diva2:409890
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Strategies to explore the membrane proteome of a cell
Open this publication in new window or tab >>Strategies to explore the membrane proteome of a cell
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope plays key roles in numerous processes such as maintaining cellular integrity, communication with other cells, signal transduction, maintenance of cellular homeostasis, and regulation of the traffic of molecules between the cell and the extracellular milieu. Essential membrane components in many of these processes are proteins. It is estimated that ~20-30% of the predicted open reading frames (ORFs) of all organisms encode membrane proteins. Furthermore, two thirds of drug targets are membrane proteins. However, despite their importance, membrane proteins have so far been mostly neglected in most proteomic studies, due to the inherent challenges in analyzing them.

The focus of this thesis is to devise strategies that allow investigation of membrane proteins and their associated complexes. Optimization of sample preparation in the underlying studies has allowed important goals to be reached in membrane protein analyses at various levels such as elucidation of their primary structure by collision-induced dissociation (CID) and electron-capture dissociation (ECD) mass spectrometry (MS), profiling membrane proteins and their complexes, the discovery of novel protein complexes, definition of their topology, and unambiguous identification of protein-bound ligand(s). This thesis paves the way for better characterization of membrane proteins and their assemblies hinting towards the crucial role(s) they play in maintaining normal cell physiology.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholm University, 2011. 94 p.
Keyword
Membrane proteins, Proteomics, Escherichia coli, Enterococcus faecalis, Synechocystis, Mass Spectrometry, BN/SDS-PAGE, FT-ICR-MS, Myelin P2 protein
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56848 (URN)978-91-7447-211-0 (ISBN)
Public defence
2011-06-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript. Available from: 2011-05-12 Created: 2011-04-28 Last updated: 2012-01-24Bibliographically approved
2. A model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803
Open this publication in new window or tab >>A model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyanobacteria constitute a unique group of eubacteria, possessing outer and plasma membranes as well as internal thylakoid membranes, the site for both photosynthesis and respiration. The combination of sucrose density centrifugation and two-phase partitioning methods leads to purification of different membranes from the cyanobacterium Synechocystis sp. PCC 6803 (referred to as Synechocystis). The standard proteomics methods, based on 1D- and 2D-gel electrophoresis, followed by protein digestion and MALDI-TOF MS, led to identification of 76 thylakoid and 51 plasma membrane proteins. In order to increase the number of identified proteins a shotgun proteomics approach was employed. Proteins were digested in complex mixtures, followed by LC separation of obtained peptides coupled to MS/MS. This approach led to identification of 379 different thylakoid and plasma membrane proteins, of which 124 were integral membrane proteins. 

The complex membrane organization of Synechocystis requires a unique system for transport and sorting of proteins into extracytosolic cell compartments. Obtained gel-based and shotgun proteomics data as well as results of the multivariate sequence analysis of both soluble and integral membrane proteins allowed to suggest a new mechanism for membrane organization and protein sorting in Synechocystis. The plasma and thylakoid membranes are proposed to be dynamically connected and both soluble and integral membrane proteins are inserted into connection points.

The Synechocystis genome possesses two genes encoding leader peptidases. Proteomic studies revealed that Sll0716 is localized in the thylakoid membrane (LepT), whereas Slr1377 in the plasma membrane (LepP). The BN/SDS-PAGE of the total membranes from mutant LepT revealed that LepT is directly involved in the processing of several photosynthetic and lumenal subunits, required for the assembly and function of PSI and maintaining of the thylakoid membrane organization in Synechocystis.

 

 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 63 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-29043 (URN)978-91-7155-917-3 (ISBN)
Public defence
2009-09-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, 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 4: In progress. Paper 5: In progress. Available from: 2009-09-08 Created: 2009-08-07 Last updated: 2011-05-03Bibliographically approved

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