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A model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Birgitta Norling)
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: urn:nbn:se:su:diva-29043ISBN: 978-91-7155-917-3 (print)OAI: oai:DiVA.org:su-29043DiVA: diva2:231481
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
List of papers
1. Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803
2005 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 18, 4905-4916 p.Article in journal (Refereed) Published
Abstract [en]

Purified thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803 were used for the first time in proteomic studies. The membranes were prepared by a combination of sucrose density centrifugation and aqueous polymer two-phase partitioning. In total, 76 different proteins were identified from 2- and 1-D gels by MALDI-TOF MS analysis. Twelve of the identified proteins have a predicted Sec/Tat signal peptide. Fourteen of the proteins were known, or predicted to be, integral membrane proteins. Among the proteins identified were subunits of the well-characterized thylakoid membrane constituents Photosystem I and II, ATP synthase, cytochrome b6f-complex, NADH dehydrogenase, and phycobilisome complex. In addition, novel thylakoid membrane proteins, both integral and peripheral were found, including enzymes involved in protein folding and pigment biosynthesis. The latter were the chlorophyll biosynthesis enzymes, light-dependent protochlorophyllide reductase and geranylgeranyl reductase as well as phytoene desaturase involved in carotenoid biosynthesis and a water-soluble carotenoid-binding protein. Interestingly, in view of the protein sorting mechanism in cyanobacteria, one of the two signal peptidases type I of Synechocystis was found in the thylakoid membrane, whereas the second one has been identified previously in the plasma membrane. Sixteen proteins are hypothetical proteins with unknown function.

Place, publisher, year, edition, pages
Weinheim: WILEY-VCH, 2005
Keyword
Gel electrophoresis, Mass spectrometry, Synechocystis 6803, Thylakoid membrane
Identifiers
urn:nbn:se:su:diva-29229 (URN)10.1002/pmic.200500111 (DOI)
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2017-12-13Bibliographically approved
2. Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins
Open this publication in new window or tab >>Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins
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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.

Keyword
cyanobacteria, integral proteins, plasma membrane, proteome, Synechocystis 6803
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56160 (URN)10.1111/j.1742-4658.2006.05624.x (DOI)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
3. Proteins in different Synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis
Open this publication in new window or tab >>Proteins in different Synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis
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2007 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 7, 2420-2434 p.Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria have a cell envelope consisting of a plasma membrane, a periplasmic space with a peptidoglycan layer, and an outer membrane. A third, separate membrane system, the intracellular thylakoid membranes, is the site for both photosynthesis and respiration. All membranes and luminal spaces have unique protein compositions, which impose an intriguing mechanism for protein sorting of extracytoplasmic proteins due to single sets of translocation protein genes. It is shown here by multivariate sequence analyses of many experimentally identified proteins in Synechocystis, that proteins routed for the different extracytosolic compartments have correspondingly different physicochemical properties in their signal peptide and mature N-terminal segments. The full-length mature sequences contain less significant information. From these multivariate, N-terminal property-profile models for proteins with single experimental localization, proteins with ambiguous localization could, to a large extent, be predicted to a defined compartment. The sequence properties involve amino acids varying especially in volume and polarizability and at certain positions in the sequence segments, in a manner typical for the various compartment classes. Potential means of the cell to recognize the property features are discussed, involving the translocation channels and two Type I signal peptidases with different cellular localization, and charge features at their membrane interfaces.

Keyword
Amino Acid Sequence, Bacterial Proteins/analysis/*chemistry, Membrane Proteins/analysis/chemistry, Membrane Transport Proteins/analysis/chemistry, Molecular Sequence Data, Multivariate Analysis, Protein Transport, Proteome/*analysis, Proteomics, Sequence Analysis; Protein, Serine Endopeptidases/analysis/chemistry, Synechocystis/*chemistry
Identifiers
urn:nbn:se:su:diva-12056 (URN)10.1021/pr0605973 (DOI)000247792300002 ()17508731 (PubMedID)
Available from: 2008-01-15 Created: 2008-01-15 Last updated: 2017-12-13Bibliographically approved
4. Model for Membrane Organization and Protein Sorting in the Cyanobacterium Synechocystis sp. PCC 6803 Inferred from Proteomics and Multivariate Sequence Analyses
Open this publication in new window or tab >>Model for Membrane Organization and Protein Sorting in the Cyanobacterium Synechocystis sp. PCC 6803 Inferred from Proteomics and Multivariate Sequence Analyses
Show others...
2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 8, 3617-3631 p.Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.

Keyword
Cyanobacteria, thylakoid membranes, membrane proteins, cellular membrane organization, intermembrane sorting
National Category
Biological Sciences
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-29231 (URN)10.1021/pr200268r (DOI)000293487900028 ()
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2017-12-13Bibliographically approved
5. Inactivation of Leader Peptidase T strongly influences the thylakoid membrane organization in the cyanobacterium Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Inactivation of Leader Peptidase T strongly influences the thylakoid membrane organization in the cyanobacterium Synechocystis sp. PCC 6803
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:su:diva-29232 (URN)
Available from: 2009-08-17 Created: 2009-08-17 Last updated: 2010-01-14Bibliographically approved

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