Abstract
Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.
Highlights
Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes
As a crucial first step toward the analysis of the composition and function of the cyanobacterial plasma membrane, a procedure was developed for its complete biochemical purification (9 –11)
Plasma membranes collected from several cyanobacterial preparations were used in producing four two-dimensional gel maps from which spots were excised and trypsinized before subjection to MALDI-TOF analysis
Summary
Bacterial and Culture Conditions—Cells of Synechocystis 6803 were grown at 30 °C under 50 mol of photons mϪ2 sϪ1 of white light in BG-11 medium [12]. The membrane fraction from 38 to 42% sucrose density was collected and diluted 3-fold with 20 mM potassium phosphate (pH 7.8) followed by a centrifugation at 187,000 ϫ g for 45 min at 4 °C. Plasma membranes containing 8 mg of protein were resuspended in 0.25 mM sucrose and 5 mM potassium phosphate (pH 7.8) at a protein concentration of 50 – 60 mg/ml. The material precipitated from 1–1.5 mg of membrane proteins was solubilized in 250 l of an electrofocusing solution containing 7 M urea, 2 M thiourea, 1% (w/v) ASB-14, 2 mM tributylphosphine, and 0.5% (v/v) immobilized pH gradient buffer, pH 3–10 (Amersham Biosciences). Measured peptides masses could be excluded if their isotopic patterns were clearly atypical or if their masses corresponded to those of trypsin autolysis products or adjacent identified proteins on the gel. The prediction of transmembrane helices in identified proteins was performed using the TMHMM program The lipoproteins were predicted using PROSITE (us.expasy.org/prosite)
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