Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells

A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100Å in diameter. No larger particles, typical of light-grown chloroplasts, were observed.     

Analysis of the thylakoid outer surface. Coupling factor is limited to unstacked membrane regions

The structure of the spinach thylakoid outer surface has been examined by deepetching, a technique which exposes the true surfaces of biological membranes by sublimination of frozen dilute buffer. The membrane surface is covered with large (150 A average diameter) and small (90 A average diameter) particles. Approximately 30% of the large particles can be removed under conditions reported to selectively remove carboxydismutase from the membrane surface. The remaining large particles can be removed only under conditions which cause a loss of coupling factor activity. When purified coupling factor is readded to membranes from which all coupling factor activity has been removed, large particles reappear, indicating that they represent coupling factor molecules. Since the number of particles and the amount of ATPase activity in the reconstituted and control membranes were the same, coupling factor molecules may be attached to specific binding sites. Analysis of antibody labeling experiments, enzyme assays, and experiments involving the unstacking and restacking of thylakoid membranes indicate that coupling factor is excluded from regions of membrane stacking (grana) and is present only in unstacked membrane regions. The exclusion of coupling factor from grana, which are known to be centers of intense photosynthetic activity, strongly suggests that the mechanism coupling electron transport to photophosphorylation is indirect. In addition to the large and small particles, in some cases regularly spaced ridges are visible on the outer surface after unstacking. Coupling factor binding sites seem to be excluded from regions where these structures occur.     

ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids)

A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids.     

THE VISUALIZATION OF THE PHOTOSYNTHETIC COUPLING FACTOR IN EMBEDDED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were stained with aqueous uranyl acetate immediately after glutaraldehyde-osmium fixation but before dehydration and embedding. Under these conditions, the lamellae are shown in thin sections to have 95-Å x 115-Å coupling factor particles on their surfaces. The particles can be seen only on the matrix side of nonopposed thylakoids, and are shown to occur on both stromal and granal lamellae, regardless of the organization of the lamellae into stacks. It is estimated that, in native, fully coupled chloroplast lamellae, there is on the average one coupling factor for every 500 chlorophyll molecules. The morphological appearance of the particles is not affected by a variety of buffers, by changes in illumination or temperature, or by alterations in the energy state of the membranes during preparation. The particles can be removed from the membranes with low concentrations of Na₂EDTA, and the photophosphorylating activity of the membranes is concomitantly lost. Both the activity and the appearance of the particles can be restored to the membranes by rebinding EDTA-extracted coupling factors to the uncoupled membranes.     

Ultrastructure of the Cell Envelope of Escherichia coli B After Freeze-Etching

The cell envelope of Escherichia coli B was investigated with the freeze-etching technique. A considerable gain in visible structural detail over more conventional electron microscopic techniques was obtained. The inner surface of the plasma membrane revealed a smooth surface sparsely studded with particles measuring from 5 to 10 nm in diameter, whereas the outer surface of the plasma membrane showed many more particles of corresponding diameter. The freeze-etched cell wall appeared to be a multilayered structure. The innermost layer could be observed as a profile studded with closely packed elements of about 10 nm in diameter. External to this layer was a smooth surface bordering the outermost cell wall layer. When frozen in the absence of glycerol the outermost surface observed in the cell wall was smooth, but when grown in the presence of glycerol it had a "wavy" appearance with small particles attached to it. The observations support current concepts on the ultrastructure of the enterobacterial cell envelope.     

Structural map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer

Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. The vesicle surface was labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine. Steady-state energy transfer between the fluorescent-labeled enzyme (energy donor) and varying concentrations of the ethanolamine derivative (energy acceptor) indicated that the distance of closest approach between the energy donor and the outer vesicle surface is 16-24 A. Two specific sites on the gamma-polypeptide were reacted with a coumarinylmaleimide derivative; one is a sulfhydryl that can be labeled only on the thylakoids under energized conditions (the "light" site), while the other is the disulfide site that regulates enzymatic activity. Energy-transfer measurements utilizing steady-state fluorescence and fluorescence lifetime methods indicated that the dicyclohexylcarbodiimide site is approximately 41 A from the light site and approximately 50 A from the gamma-disulfide site. These distances are used to extend the current structural model of the chloroplast coupling factor.     

On the differences of molecular aggregates structure with long wave fluorescence in the photosystems I and II]

Galactolipase and protenase action on the low temperature fluorescence spectra of light chloroplast fragments obtained from grana or intergrana thylakoids and grana thylakoids system before and after isolation photosystem I particles have been studied. Identical hydrolytic enzymes action in the two type photosystem I particles have been studied. Identical hydrolytic enzymes action in the two type photosystem I particles were observed. Grana thylakoids system after removing photosystem I particles contained photosystem II in the most purified form. These measurements results confirmed our previous suggestion that the band at 735 nm in the low temperature fluorescence spectra of light and heavy fragments belongs to the different native chlorophyll a aggregates.     

Light-dependent cleavage of the gamma subunit of coupling factor 1 by trypsin causes activation of Mg2+-ATPase activity and uncoupling of photophosphorylation in spinach chloroplasts

Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.     

Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.     

Intact Chloroplasts Show Ca-Gated Switching between Localized and Delocalized Proton Gradient Energy Coupling (ATP Formation)

Intact chloroplasts were compared to isolated thylakoids as to whether storage of the organelle in high KCl medium caused the energy coupling reactions to show a delocalized or a localized proton gradient energy coupling response. With isolated thylakoids, the occurrence of one or the other energy coupling mode can be reversibly controlled by the concentration of mono- and divalent cations used for the thylakoid storage media. Calcium was shown to be the key ion and previous evidence suggested a Ca²⁺-controlled gating of H⁺ fluxes in the thylakoid membrane system (G Chiang, RA Dilley [1987] Biochemistry 26: 4911-4916). Isolated, intact chloroplasts, which retained the outer envelope membranes during the 30 min or longer storage treatments in various concentrations of KCl and CaCl₂ (with sorbitol to maintain iso-osmotic conditions), were osmotically burst in a reaction cuvette and within 3 minutes were assayed for either a localized or a delocalized proton gradient energy coupling (ATP formation) mode. The intact chloroplast system was analogous to isolated thylakoids, with regard to the effects of KCl and CaCl₂ on the energy coupling mode. For example, adding 100 millimolar KCl to the intact organelle storage medium resulted in the subsequent ATP formation assay showing delocalized proton gradient coupling just as with isolated thylakoids. Adding 5 millimolar CaCl₂ to the 100 millimolar KCl storage medium resulted in a localized proton gradient coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations and testing the energy coupling response showed that the stromal milieu has enough Ca²⁺ to cause the localized coupling response even though there was about 80 millimolar K⁺ in the intact chloroplasts used in this study (determined by atomic absorption spectrophotometry). Extrapolating the intact chloroplast data to the whole leaf level, we suggest that proton gradient energy coupling is normally of the localized mode, but under certain conditions it could be either localized or delocalized, depending on factors that affect the putative Ca²⁺-regulated proton flux gating function.     

Fine Structures of Cell Envelopes of Chlamydia Organisms as Revealed by Freeze-Etching and Negative Staining Techniques

The cell walls of Chlamydia psittaci (meningopneumonitis strain) were examined by the freeze-etching and negative staining techniques. It was observed that the cleaved convex surface of the developmental, reticulate body was covered with numerous non-etchable particles 9 to 10 nm in diameter, these particles being rarely seen on the concave surface. Similarly, the convex surface of the mature, elementary body (EB) was covered with many particles but the concavity lacked these particles. After etching, the smooth concave surface of the EB appeared to have a hexagonally arrayed subunit structure, on which the button structure (B structure) was observed. Each B structure had a diameter of 27 nm and several B structures were grouped together in a hexagonal arrangement with a center-to-center spacing of 45 nm. In a limited area of the negatively stained EB cell wall, hexagonally arrayed rosette structures were present, with a center-to-center spacing similar to the B structures seen in the freeze-etched preparation. Each rosette, about 19 to 20 nm in diameter, appeared to be composed of a radial arrangement of nine subunits. The freeze-fractured cell wall-cytoplasmic membrane complexes indicated that the outer surface of the cytoplasmic membrane which appeared as the convex surface was covered with the fine particles, and thus it was likely that frozen EB was cleaved at the gap between the cell wall and ctyoplasmic membrane. On the cleaved inclusion, several groups of fine particles were observed. In each group, the particles were arranged hexagonally with the spacing ranging from 20 to 50 nm.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.     

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.     

Structure des membranes biologiques: localisation des galactosyldiglycerides dans les chloroplastes au moyen des anticorps specifiques. II. Etude en microscopie electronique a l'aide d'un marquage a la peroxydase

Structures of biological membranes: Localisation of galoctosyldiglycerides in chloroplasts by means of specific antibodiesII. Treatment with peroxidase: electron microscope studyThe results obtained in this study confirm that the outer membrane of the chloroplast envelope contains galactosyldiglycerides and that these molecules are uniformly distributed over the membrane surface. The results show that the galactosyldiglyceride molecules that are accessible to antibodies at the level of the thylakoids are distributed over the membrane surface in discrete patches.