Effects of Photoperiod on Supramolecular Architecture of Thylakoid Membranes from a Rice Mutant (Oryza sativa Nongken 58S)

Plants of a rice mutant (Hubei photoperiod-sensitive genic male-sterile rice, Oryza sativa L. Nongken 58S) and its wild type cv. Nongken 58 were cultured in natural summer conditions in Beijing. After induction of proper photoperiods small panicle at the stem tip emerged and developed to the stage of secondary rachis-branch and spikelet primordium formation. Subsequently, part of the rice plants received long day (LD), i.e. 10 h of day-light treatment followed by 5 h of white fluorescent illumination with 1～2 Wm-2) . The others were exposed to daylight for 10 h alternating with a 14 h of dark period as short day (SD) treatment. After 10 days of the photoperiodic treatments, the chloroplast ultrastructure of the first leave below the flag leaf was examined by freeze-fracture rotary and unidirectionally shadowed electron microscopy. At anthesis stage, Nongken 58S plants with LD treatment showed complete pollen sterility, while the same plants with SD treatment exhibited normal fertility. And fertility of Nongken 58 was not affected by photoperiod treatments. The results from electron microscopic observation showed no significant effects of either SD or LD treatment on the freeze-fractured uhrastructure of thylakoid membranes in Nongken 58. No significant difference in particle density and size distribution was found on stacked and unstacked thylakoid membrane regions of the Nongken 58S-SD and those of Nongken 58 rice. However, the particle density of the endoplasmic fracture face in the staked region (EFs) and protoplasmic fracture face in the staked region (PFs) faces detected from the leaf thylakoid membranes of Nongken 58S-SD rice was significantly higher than that of the corresponding faces from Nongken 58S-LD. In some cases much more particles on EFs faces of thylakoid membranes isolated from Nongken 58S-SD rice appeared as paracrystalline particle array, indicating increases in the number of PS Ⅱ reaction centres, LHC I and Cyt b6/f per unit area of thylakoid membrane. The particle density of the endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces from unstacked thylakoid membranes of Nongken 58S-LD was less than that of the corresponding faces from Nongken 58S-SD. And the particle density of PFu faces from margin and end of the membranes of the grana thylakoids of LD-treated Nongken 58S leaves was also less than that of unstacked thylakoid membranes from SDtreated rice. In severe cases, most of the particles on endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces were even missing, indicating a decrease in the numbers of photosystem Ⅰ , LHCⅠ , Cyt b6/f and ATPase per unit area of' thylakoid membrane. The above results could further provide an augmentation for explaning the photoperiod-sensitive genic male-sterility.     

Ultrastructure of cell wall and thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus under the influence of temperature shifts

The ultrastructure of the cell wall and the thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus was studied by freezefracture electron microscopy after temperature shifts. Different fracture faces of the outer, the cytoplasmic and the thylakoid membranes were demonstrated when the preparation-temperature was in the range of the optimal growth temperature at 52°C or after fixation at 52°C. In the outer membrane of the cell wall two fracture faces with holes and 7.5 nm intramembrane particles were detected. On both the outer (EF) and inner (PF) leaflet of the cytoplasmic membrane randomly distributed particles were demonstrated. The particle density on the PF-face was approx. three times that of the EF-face. The EF-face of the thylakoid membrane exposed rows of particles with an average diameter of 10 nm. The spacing between the particle rows was 35–50 nm. This regular particle arrangement on the EF-face was demonstrated only in a few cases. Mostly the intramembrane particles were distributed randomly on the thylakoid fracture faces. The particle density of thylakoids with a random distribution was approx. in the same range both on the EF-and PF-face. The EF-particles fall into four groups of 9,10,11, and 12.5 nm. The main particle class was the 10 nm class. The PF-face exposed smaller particles with two maxima at 8.5–9 nm and 10 nm. When Synechococcus lividus OH-53s was chilled to temperatures below 30–35°C before the freeze-etch preparation a phase transition took place after the temperature shift. On the fracture faces of the thylakoid and cytoplasmic membranes particle depleted areas occurred. The size of the areas were different in both membranes and dependent on the velocity of cooling. Contrary to Synechococcus lividus OH-53s in the mesophilic Synechococcus strain 6910 the phase transition point was 15°C. The lower phase transition point may be due to a higher content of unsaturated fatty acids.Dedicated to Prof. D. Peters (Hamburg) on the occasion of the 65th anniversary of his birthday     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.     

Microsomal T system: a stereological analysis of purified microsomes derived from normal and dystrophic skeletal muscle

Heterogeneous populations of microsomes obtained from normal and dystrophic chicken pectoralis muscle were separated into two subfractions by an iterative loading technique. The buoyant density of the sarcoplasmic reticulum (SR) microsomes was increased after loading them with calcium oxalate. Several incubations in the transport medium were necessary to load all of the SR. The fraction that did not form a pellet contained microsomes which displayed freeze-fracture faces that had a low density of particles. A stereological analysis was used on membrane fracture faces of intact muscle to generate reference particle density distributions, which were compared with the distributions measured on the microsomal fracture faces. The concave microsomal fracture faces of purified microsomes which did not load calcium oxalate had particle distributions nearly identical to the distributions of intact P-face T tubules. The morphological data suggest that this subfraction is microsomal T system. Biochemical measurements show negligible amounts of specific Na+, K+-ATPase activity, suggesting that there was little contamination from the surface membrane in this subfraction. Furthermore, an active Ca2+-ATPase is demonstrated in both normal and dystrophic T-tubular membranes.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.     

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.     

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.     

Freeze-fracture study of heart mitochondria in the condensed or orthodox state

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.     

Freeze-fracture study of heart mitochondria in the condensed or orthodox state

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.     

Topographical relations of intramembrane particle distribution patterns in human sperm membranes

The distribution of intramembrane particles in human sperm membranes has been explored with particular reference to the topographical region of the sperm cell and the membranes' fracture face. Conspicuous differences in the size, arrangement, density, and lateral mobility of intramembrane particles between some topographically distinct membrane domains are demonstrated. The greatest regionality is exhibited by the plasma membrane. In sperm head regions, it shows a significant variability and changes its particle distribution during culture in capacitating medium. In contrast, little variability and no changes during the incubation are seen in the acrosomal and nuclear membranes. Striking is the difference in particle distribution on the E face of the outer acrosomal membrane between the acrosomal and equatorial regions. It is suggested that the invariable regional difference in the organization of the outer acrosomal membrane may bear on the different behavior of its two main domains during sperm capacitation and acrosome reaction.     

The Supramolecular Architecture in Chloroplast Thylakoid Membranes of Warigal Wheat

Thylakoid membranes of chloroplast from first leaf and flag leaf of wheat Warigal were examined by freeze-fracture and rotary shadowing etectron microscopy. The shape, size, density and size distribution of freeze-fracture partieles of their four faces were measured and plotted as three-dimensional histograms by a Hewlett-packard 9874 A digitizer with a HP 9845 B Computer and HP 9872 C plotter. When comparisons were made among different fracture faces and between the corresponding faces of the first leaf and the flag leaf, we found that the supramolecular architecture on the four fracture faces of the flag leaf differs from that on the corresponding faces of the chloroplast thytakoid membranes of the first leaf. The most significant difference was that the EFs particles contain the photosystem Ⅱ reaction centres associated with LHCP and the PFs particles were mostly light-harvesting complex. There was a 15% increase in EFs particle density, a 22% increase in PFs particle density and a 28% increase in EFu particle density. The large PFu particles contained the photosystem Ⅰ reaction centre and the flag lcaves contained 5% more than the first leaves. In addition, the stacking of thylakoid membranes in the flag leaf was 5% more than those in the first leaf.Thus, it provides theoretical basis for the fact that the flag leaf has higher photosynthetic rate.     

A COMPLEMENTARY FREEZE-FRACTURE STUDY OF CHRYSOCHROMULINA CHITON (HAPTOPHYCEAE)

Complementary freeze-fracture replicas and high resolution tantalum-tungsten shadowing have been used in a study of the membanes of the marine alga Chrysochromulina chiton. Membrane particle populations range from 38/100 nm² in the plastid to 2/100 nm² in the pyrenoid cap membrane. Membrane asymmetry was evident in all membranes, but was most obvious in those with higher particle numbers. In all complementary replica pairs, particles were always more numerous on protoplasmic fracture faces. Small, particle-free areas with bordering particles were also seen as recurring membrane features. Complementarity of matching fracture faces was seen for very small background granularity patterns and for large membrane components, but not for particles. Complementarity can also be seen in non-membranous fracture faces both within and external to the cell, suggesting the presence of polymeric materials in these areas that produce “particles” due to plastic deformation.     

The Supramolecular Organization of Photosynthetic Membranes of the Thallus Stage of Porphyra leucosticta Thuret. (Bangiales,Rhodophyta) Visualized by Freeze-Fracture

The supramolecular organization of the thylakoid membranes of the thallus stage in the red alga Porphyra leucosticta is studied in replicas of rapidly frozen and fractured cells. Freeze-fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF), because the lamellae in red algal chloroplasts are not stacked. The PF reveals numerous, tightly packed, but randomly distributed particles (density range from 2970 to 3550 particles/μm²). In contrast, the EF particles appear organized into parallel rows, the spacing of which is about 60–70 nm (about 8–9 particles occur along 100 nm of the line that is formed). Significant numbers of single EF particles are randomly distributed between the EF particle rows. The particles on both fracture faces (PF and EF) fall into two size classes: 10 to 11 nm (major size class) and 14 to 15 nm (minor size class).     

Quantitation of particles in the freeze-fractured nuclear membrane after renal ischemia.

Changes in the number and sizes of membrane-associated particles have been quantitated in the protoplasmic (P) and exoplasmic (E) fracture faces of the outer membrane of nuclei isolated from the inner cortex following renal ischemia and reflow in the rat. No changes were observed in the inner nuclear membrane. After 20-min ischemia, the number of particles in both fracture faces decreased. With reflow, the total number of particles decreased after both 20- and 60-min ischemia. The partition coefficient (Kp = CPF/CEF) increased from 10 to 11 and 17 at 20- and 60-min ischemia then fell below control values to a Kp of 7 after 120 min. After reflow, Kp steadily decreased except after 20-min ischemia followed by 240-min reflow when Kp began to rise. The sizes of particles were predominantly 60 A in the P face of control outer membranes but became larger after ischemia. After 20- and 60-min ischemia with reflow, the size distribution became more normal. The shifts in particle numbers and sizes seem to indicate modifications within the membrane resulting from ischemia.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W 3110. While these proteins were distributed randomly at the growth temperature (37 °C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 °C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes.The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction.Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while d-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes.These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.     

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.