Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium Limicola

The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 × 70 nm to 100 × 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5–6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40° and 60° with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles.The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl a-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl a. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.     

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl₂ was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.Abbreviations Chl chlorophyll - DCPIP dichlorophenolindophenol - DPC diphenolcarbazide - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane     

A rapid reverse phase evaporation method for the reconstitution of uncharged thylakoid membrane lipids that resist hydration

Comparison of several lipid reconstitution methods showed that they were not equally efficient at transferring the predominant thylakoid lipid, monogalactosyldiglyceride (MG), to the aqueous phase. We report a reverse phase evaporation method that employs Freon 11 as a lipid solvent and is capable of successfully hydrating MG in spinach (Spinacia oleracea L.) at room temperature within minutes. Using this method it is possible to force an equal weight mixture of MG and digalactosyldiglyceride into small bilayer vesicles without the formation of inverted micellar `lipidic particles' in the membranes.     

Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the expolasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 Å. The corresponding histogram for heterocyst thylakoids lacks the 100 Å size class, but has a very large peak at about 55 Å with a shoulder at 75 Å. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 Å for vegetative cells and 64 Å for heterocysts. The thylakoids of both cell types have about 5600 particles/μm² on the P-face. On the E-face, the density drops from 939 particles/μm² on vegetative cell thylakoids to 715 particles/μm² on heterocyst thylakoids. The data suggest that the 100 Å E-face particle of vegetative cell thylakoids is a PSII complex. The 55 Å EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 Å EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.