RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

THE MOLECULAR ORGANIZATION OF CHLOROPLAST MEMBRANES

The presence of subunits in chloroplast membranes is suggested by polarization, fluorescence, and X-ray studies. Subunits (quantasomes) may be observed in the electron microscope on dried shadowed membranes and in replicas of membranes produced by the freeze-etching technique. Regular subunits are also observed with the electron microscope in thin sections of chloroplast membranes. Chemical considerations suggest that many membranes are composed of lipoprotein subunits. Thin sections reveal two types of chloroplast membranes, the fret membranes composed of one layer of subunits, and the partitions composed of two layers of subunits. Chloroplast membranes consist of about 45% protein and 55% lipid. Some 80% of the lipids are the highly surfactant glycolipids. In this paper the subunits are visualized as assymetric lipoproteins, probably having a protein core surrounded by components determined by the nature and environment of the membrane. Since the stroma, fret channels, and loculi contain aqueous materials, it is further postulated that the membranes bordering these spaces bind the highly surfactant glycolipids. The region between the two rows of subunits in the partition appears to be highly hydrophobic, rich in chlorophyll, and low in glycolipids. Some chlorophyll also may occur within the subunits both in the partitions and in the fret membranes. Since four subunits appear to comprise a quantasome, at least two types of forces, inter- and intra-quantasome forces, bind the subunits together in sheets. Chloroplast membranes thus differ from a “unit membrane” in two important respects: (1) they must be an aggregate of globular subunits, and (2) the lipoprotein subunits consist of a protein matrix which binds the chlorophylls and lipids by hydrophobic association with their hydrocarbon moieties.     

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.     

MEMBRANES OF MESOPHYLL CELLS OF NICOTIANA RUSTICA AND PHASEOLUS VULGARIS WITH PARTICULAR REFERENCE TO THE CHLOROPLAST

Weier , T. E., and W. W. Thomson . (U. California, Davis.) Membranes of mesophyll cells of Nicotiana rustica and Phaseolus vulgaris with particular reference to the chloroplast. Amer. Jour. Bot. 49(8): 807–820. Illus. 1962.—The endoplasmic reticulum in mesophyll cells is represented by short lengths of irregularly disposed, paired membranes. It is occasionally associated with a typically double nuclear envelope. Groups of irregularly parallel, paired membranes suggesting disorganized dictyosomes occur infrequently. Mitochondria are unevenly distributed in mesophyll; they are large and have sparse tubular cristae around their periphery. In the great majority of instances the bounding membrane is diffusely stained with KMnO₄. When it is sharp and distinct, it may be double as usually pictured, or it may have well-delineated stretches of a single membrane bounding 25–50% of its circumference. The tonoplast and ectoplast are very fragile, the former appearing as a single dark line. In young leaves the ectoplast is visualized as a continuous single membrane adjacent to the cell wall, but in our micrographs of mature leaves it is always discontinuous. The plastid membrane sometimes is distinctly double, having 2 dark components bounding a light component. In the great majority of cases, however, this membrane is either a solid dark line, or the clear component of the double membrane is crossed by delicate dark lines giving the membrane a braided, or scalariform appearance. The various appearances of the membrane may intergrade with each other. The width of the plastid membrane is variable, ranging from 200 to 400 A. The inner component may invaginate into the stroma, and bodies may form in the clear space between the 2 outer membrane components. Micrographs suggest that these bodies, and others formed by small masses of stroma, may be expelled into the hyaloplasm, where they exist as spherical single-membraned particulates. The reality of the variable structure of the plastid membrane is discussed in light of concepts of membrane activity, molecular structure, and the relation of these factors to possible artifacts.     

FREEZE-FRACTURED CHLOROPLAST MEMBRANES OF GONYAULAX POLYEDRA (PYRROPHYTA)1

The chloroplast membranes of Gonyaulax polyedra Stein were studied in replicas of rapidly frozen and fractured cells. The thylakoid EF_s face lacked the large 15–16 nm particles characteristic of plants with the light-harvesting chlorophyll a/b protein, presumably because the principal light-harvesting protein of Gonyaulax is the small water-soluble peridinin-chlorophyll-protein and the chlorophyll a/b protein is absent. As in other plants, the EF_s thylakoid fracture face carried more particles (4 ×) than EF_uface. The PF faces of the thylakoid showed twice as many particles as did the EF_s faces. No circadian differences in the number or size of thylakoid membrane particles could be detected. Three membranes comprise the chloroplast envelope in Gonyaulax. They could be clearly differentiated in freeze-fractured cells. The middle envelope membrane carried many fewer particles on both the EF and PF faces than did the other two envelope membranes. The PF faces of both the outer and inner envelope membranes showed more particles than the EF faces, as do many other membranes which have been examined.     

细胞色素f在光合膜上功能的初步探讨

用阳离子一竭尽的菠菜叶绿体膜作为实验材料,比较研究了胰蛋白酶消化的叶绿体膜和非消化的叶绿体膜的四阶导数吸收光谱、细胞色素f的还原减氧化差异光谱和电子传递速率的变化,试图探索细胞色素f在光合膜上的功能.主要结果于下:(1)胰蛋白酶消化引起叶绿体膜四阶导数吸收光谱位于554nm吸收峰(CYT.f的特征吸收峰)下降和细胞色素含量的明显减少.说明胰蛋白酶损伤细胞色素f.(2)胰蛋白酶消化对叶绿体膜从DCIPH_2到甲基紫精的非循环式电子传递速率没有影响,而对PMS存在下的甲基紫精光还原速率有明显的刺激作用.说明胰蛋白酶损伤与循环式电子流有关的细胞色素f.根据试验结果的分析.我们推测,在光合膜上可能存在两种类型的细胞色素f.一种可能与循环式电子流有关.另一种可能与非循环式电子流有关.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.     

POLYSOMES OF THE SEA URCHIN EMBRYO: AN IMPROVED METHOD FOR EXTRACTION OF INTEGRATED POLYSOMES

Suitable conditions for extracting integrated polysomes from embryos of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus were investigated.
Integrated polysomes could not be extracted under the conditions reported by other investigators. It was found, however, that use of 5 m m MgCl₂, 0.30 m KCl, 0.5 m m EDTA and 2 m m cycloheximide was effective for maintaining the integrity of polysomes. At higher concentrations of Mg²⁺, and even at higher concentrations of K⁺, monosomes and polysomes aggregated to form polysome-like particles which had sedimentation patterns with a small amount of nascent peptide. Thus, a medium consisting of 0.05 m Tris-HCl buffer, pH 7.5, 0.30 m KCl. 5 m m MgCl₂, 0.5 m m EDTA-2K, 2 m m cycloheximide, 5 m m mercaptoethanol and 0.5% (v/v) Nonidet P-40 is concluded to be the most suitable for extraction of sea urchin polysomes. Under the conditions used EDTA did not suppress polysome degradation completely and their degradation was linear with time.     

STRUCTURAL DIFFERENTIATION OF STACKED AND UNSTACKED CHLOROPLAST MEMBRANES : Freeze-Etch Electron Microscopy of Wild-Type and Mutant Strains of Chlamydomonas

Wild-type chloroplast membranes from Chlamydomonas reinhardi exhibit four faces in freeze-etchreplicas: the complementary Bs and Cs faces are found where the membranes are stacked together; the complementary Bu and Cu faces are found in unstacked membranes. The Bs face carries a dense population of regularly spaced particles containing the large, 160 ± 10 A particles that appear to be unique to chloroplast membranes. Under certain growth conditions, membrane stacking does not occur in the ac-5 strain. When isolated, these membranes remain unstacked, exhibit only Bu and Cu faces, and retain the ability to carry out normal photosynthesis. Membrane stacking is also absent in the ac-31 strain, and, when isolated in a low-salt medium, these membranes remain unstacked and exhibit only Bu and Cu faces. When isolated in a high-salt medium, however, they stack normally, and Bs and Cs faces are produced by this in vitro stacking process. We conclude that certain particle distributions in the chloroplast membrane are created as a consequence of the stacking process, and that the ability of membranes to stack can be modified both by gene mutation and by the ionic environment in which the membranes are found.     

THE NUCLEAR ENVELOPE : ITS STRUCTURE AND RELATION TO CYTOPLASMIC MEMBRANES

An electron microscope study of thin sections of interphase cells has revealed the following:— Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.     

铜对类囊体膜叶绿素-蛋白质复合物组成和光谱特性的影响

菠菜和青菜类囊体膜经SDS-PAGE*可分别分离出8条和7条含叶绿素的区带。经Cu螯合剂处理后发现菠菜CPIa、青菜CPI_a、LHCP_2带缺失,菠菜CPIa_1 LHCP_2和青菜CPI减少,两者的LHCP_3明显增加。外源Cu(5mmol/L CuCl_2)可使菠菜CPa_1带缺失。青菜CP_a带缺失,并且使菠菜CPI带的吸收峰由678nm移到672nm,在652nm处有一微弱小肩,并且出现679nm荧光发射峰,表现出LHC-Ⅱ的某些光谱特性。同时使菠菜和青菜的LHCP_1和LHCP_2的吸收峰均由672nm分别移到668nm和669nm,并且使LHCP_2在724nm处产生较强的荧光发射,接近于LHC-Ⅰ的某些光谱特性。由此初步认为,铜可能通过变构作用来调节两个光系统间从作用中心到捕光色素蛋白复合物,以及二者捕光色素蛋白复合物本身之间的能量转移。     

PERYLENE ATTACHED TO DNA THROUGH STIFF OR FLEXIBLE LINKER: DUPLEX STABILITY AND FRET

Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2′-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2′-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.     

THE USE OF MICROBIAL MEMBRANES TO ACHIEVE ANAEROBIOSIS

Several years ago, it was observed that sterile microbial membrane preparations stimulated recovery of certain radiation-injured bacteria. Later it was noted that these same preparations reduce dissolved oxygen to water in a variety of environments, including bacteriological media. This reduction of oxygen is an enzymatic process and is influenced by parameters such as temperature, pH, and the availability of specific oxidizable substrates. Oxygenreducing membrane preparations can be made from several different bacterial species. When added to liquid or solid bacteriological media, membrane preparations rapidly produce and maintain anaerobic conditions favorable for the growth of a wide variety of oxygen-sensitive microorganisms. When used with a specifically designed disposable dish, membrane preparations allow the development of colonies of many anaerobic microorganisms on the surface of agar without the use of anaerobic hoods or other devices. In addition to providing conditions suitable for the growth of anaerobes, membrane preparations stimulate recovery of heat and cold injured bacteria of several different genera including facultative organisms. These results are reminiscent of the early observations regarding the recovery of radiation-injured bacteria. In addition to their usefulness in microbiology, oxygen-reducing membrane preparations have the potential for protecting a wide variety of oxygen-sensitive organic compounds.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

A MAJOR POLYPEPTIDE OF CHLOROPLAST MEMBRANES OF CHLAMYDOMONAS REINHARDI : Evidence for Synthesis in the Cytoplasm as a Soluble Component

Electrophoresis of thylakoid membrane polypeptides from Chlamydomonas reinhardi revealed two major polypeptide fractions. But electrophoresis of the total protein of green cells showed that these membrane polypeptides were not major components of the cell. However, a polypeptide fraction whose characteristics are those of fraction c (a designation used for reference in this paper), one of the two major polypeptides of thylakoid membranes, was resolved in the electrophoretic pattern of total protein of green cells. This polypeptide could not be detected in dark-grown, etiolated cells. Synthesis of the polypeptide occurred during greening of etiolated cells exposed to light. When chloramphenicol (final concentration, 200 µg/ml) was added to the medium during greening to inhibit chloroplastic protein synthesis, synthesis of chlorophyll and formation of thylakoid membranes were also inhibited to an extent resulting in levels of chlorophyll and membranes 20–25% of those found in control cells. However, synthesis of fraction c was not affected by the drug. This polypeptide appeared in the soluble fraction of the cell under these conditions, indicating that this protein was synthesized in the cytoplasm as a soluble component. When normally greening cells were transferred from light to dark, synthesis of the major membrane polypeptides decreased. Also, it was found that synthesis of both subunits of ribulose 1, 5-diphosphate carboxylase was inhibited by chloramphenicol, and that synthesis of this enzyme stopped when cells were transferred from light to dark.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : V. A Radioautographic Study of Membrane Growth in a Mutant of Chlamydomonas reinhardi y-1

The development of photosynthetic lamellae during greening of dark-grown Chlamydomonas y-1 cells was investigated by radioautography. Acetate-³H was used as a marker for membrane lipids. In short pulse-labeling experiments, about 50–60% of the radioactivity incorporated was found in the lipid fraction and about 25–50% in starch granules present in the chloroplast of these algae. The relative specificity of acetate-³H used as a marker for membranes was artificially increased through quantitative removal of the starch granules from fixed cells by amylase treatment. Analysis of turnover coefficients of different membrane constituents and of the contribution of turnover and net synthesis to the total label incorporated in pulse experiments indicated that the incorporation of acetate into specific lipids was mainly due to net synthesis. The distribution of radioactivity in the different lipid constituents at the end of a short pulse and after 30- and 60-min chases indicated that transacylation is minimal and may be disregarded as a possible cause of randomization of the label. Statistical analysis of radioautographic grain distribution and measurements of different structural parameters indicate that (a) the chloroplast volume and surface remain constant during the process, whereas the growth of the photosynthetic lamellae parallels the increase in chlorophyll; (b) the lamellae do not develop from the chloroplast envelope or from the tubular system of the pyrenoid; (c) all the lamellae grow by incorporation of new material within preexisting structures; (d) different types of lamellae grow at different rates. The pyrenoid tubular system develops faster than the thylakoids, and single thylakoids develop about twice as fast as those which are paired or fused to grana. It is concluded that growth of the membranes occurs by a mechanism of random intussusception of molecular complexes within different types of preexisting membranes.