Translocation of proteins across the multiple membranes of complex plastids.

Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.

Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzon merolae.

The behavior of cell nuclei, mitochondria and chloroplasts was followed during the mitotic cycle in the primitive unicellular red alga Cyanidioschyzon merolae by epifluorescence microscopy after staining with 4',6-diamidino-2-phenylindole (DAPI) or 3,3'-dihexyloxacarbocyanine iodide (DIOC6), by fluorimetry using a video-intensified microscope photon-counting system (VIMPCS), and by electron microscopy. The cells were classified into five types based on differences in the shape, size, and distribution of organelles in each cell: type I, II, III, IV, and V. Types II, III, IV, and V appear in that order concomitant with a decrease in type I after the cells are subjected to darkness during synchronous culture, suggesting that the organelles divide in the following sequence: chloroplast, mitochondrion, and cell nucleus. The mitochondrial DNA (mt-DNA) and the chloroplast DNA (cp-DNA) are synthesized during stage I, while the cell-nuclear DNA (cn-DNA) is duplicated in stage II. The mitochondria- and chloroplast-nuclear divisions are completed in stage IV. The mitochondrial and chloroplast divisions begin simultaneously in stage II, and chloroplast division finishes just prior to mitochondrial division. The end result is that C. merolae multiplies by binary fission. Electron microscopic examination of serial thin sections of the chloroplast clearly show that the chloroplasts in types II and III are divided by a chloroplast dividing ring (CD-ring), which is located on the cytoplasmic side of the outer envelope at the equatorial region between the daughter chloroplasts. The CD-ring may be involved in chloroplast division throughout the plant kingdom in primitive unicellular algae and higher plants.

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.

Photocontrol and processing of LHCP II apoprotein in Euglena: possible role of Golgi and other cytoplasmic sites.

Like other green photosynthetic eukaryotes, cells of Euglena gracilis var. bacillaris and strain Z contain a light-harvesting chlorophyll a/b complex associated with photosystem II. In Euglena, the formation of the 26.5 kDa principal light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) has a number of unusual features. The precursors to LHCP II are large polyproteins containing multiple copies of LHCP II, and photocontrol of their formation is largely translational. Under conditions favoring LHCP II accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCP II passes through the Golgi on the way to the thylakoids. The compartmentalized osmiophilic structure (COS) also shows an immunoreaction. These observations, and other discussed in this paper, suggest that light permits translation of polyprotein LHCP II precursors on cytoplasmic ribosomes of the rough endoplasmic reticulum (ER) and that these pass through the ER to the Golgi where, presumably, further modifications take place. Since an LHCP II immunoreaction is found in Golgi vesicles, these may transport the nascent LHCP II to the plastid and facilitate its uptake.

[Immunocytochemical studies on the behavior photosynthetic enzymes during the cell cycle of synchronized cells of Euglena gracilis Z].

Euglena cells were grown synchronously under photoautotrophic culture conditions on a 14 h light-10 h dark alternations. Changes in morphology of the pyrenoid and those in distribution of RuBisCO in chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. The immunoreactive protein were densely localized in the pyrenoid, and thinly distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. From a comparison of photosynthetic CO2 -fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO2 -fixation in photosynthesis. Cells of Euglena contain a LHC II. The precursors to LHC II are large polyproteins containing multiple copies of LHC II, and photocontrol of their formation is largely translational. Under conditions favoring LHC II accumulation in the thylakoids, a reaction with anti-LHC II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHC II passes through the Golgi on the way to the thylakoids.

Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures. IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division.

Cells of Euglena gracilis were synchronized by applying a 14-h light:10-h dark regimen under photoautotrophic conditions and a 10-h light:14-h dark regimen under photoorganotrophic conditions. At a stage just prior to chloroplast division in the cell cycle of these synchronized cultures, chloroplasts temporarily gathered in the posterior part of the cell and were connected to each other by many bridges. Part of the chloroplast aggregate surrounded about half of the nuclear surface, making connections or close contacts at many sites. A chromosome was always attached to the inner membrane of the nuclear envelope at the site of association with the chloroplast. The nucleoids in these aggregate chloroplasts, examined by staining with 4',6-diamidino-2-phenylindole, a DNA fluorochrome, showed profiles of strings or strands with branchings, under photoorganotrophic conditions at least, and some parts of the branchings came close to the site of association with the nucleus. The association between the chloroplast aggregate and the nucleus was also observed in Euglena cells placed in continuous darkness after synchronization under photoorganotrophic conditions, suggesting that these organellar associations are related to the Euglena cell cycle but are not the result of light:dark alternations used for cell synchronization.

Behavior of mitochondria in synchronized cells of Chlamydomonas reinhardtii (Chlorophyta).

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12 hour:12 hour light:dark regimen. Behavior of mitochondria in these cells was studied by fluorescence microscopy using a mitochondrial membrane-binding fluorescent dye, dimethylaminostyrylmethyl-pyridiniumiodine (DASMPI), as well as by electron microscopy. Following time courses of change in frequency of occurrence of five typical morphologies of mitochondria in synchronized cells, strikingly dynamic behavior of mitochondria was demonstrated. The five types are (A) a giant global mitochondrion with large matrix and peripherally localized cristae, a part of which is in close contact with the nucleus, (B) a mitochondrion composed of thick-corded bodies connected to each other, a part of which is in contact with the nucleus, (C) thin-corded forms with a few branches, (D) small lump forms scattered in the cytoplasm, and (E) stringy forms with intricate branchings extended throughout the cytoplasm. During the early half of the light period, changes of C-->B-->C-->D occur, while the inversely sequential changes of D-->C-->B-->C proceed during the later half of the light period. The appearance of the B-type mitochondrion is accompanied by a transient decrease of O2-uptake activity of cells. The early appearing B-type mitochondrion is temporarily turned into a giant A-type mitochondrion, concomitant with discharge of membranes into the cytoplasm and their retake by the A-type form in the process of reversion to B-type. In the reversion process, partitioning membranes are also formed in the large matrix of A-type mitochondrion.(ABSTRACT TRUNCATED AT 250 WORDS)

W10BSmL, a mutant of Euglena gracilis var. bacillaris lacking plastids.

Organized proplastid structures are absent from dark-grown and light-grown cells of Euglena gracilis Klebs var. bacillaris Cori mutant W10BSmL, based on electron micrographs of serial sections of entire cells. Fluorescence due to normal plastid DNA is undetectable in these cells after treatment with the DNA fluorochrome 4'6-diamidino-2-phenylindole (DAPI). Serial sections through a newly described compartmentalized osmiophilic structure in Euglena cells are presented.

Immunogold localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in developing proplastids of dark-grown wax-rich cells of Euglena gracilis.

Wax-rich cells of Euglena gracilis Z grown in the dark without agitation were subjected to freeze-substitution procedures in electron microscopy, which gave intact images of wax accumulated as globules in the cytoplasm. The proplastids in these wax-rich cells were shown to contain no extensive internal structures. When these cells were transferred to an inorganic medium containing ammonium salt and aerated in the dark the specific activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) increased concurrently with the development of a prolamellar body and a rudimentary pyrenoid. When cell sections were labeled with antisera prepared against RuBisCO subunits followed by protein A-gold, gold particles (RuBisCO) were localized in a narrow peripheral region between the plastid envelope and the prolamellar body during an earlier phase of the dark incubation of cells. Subsequently, immunogold particles were concentrated over the rudimentary pyrenoid formed at a site adjacent to the prolamellar body, while the stroma was only slightly labeled with gold particles. It was postulated that the small subunits of RuBisCO made in the cytoplasm combined with the plastid-made large subunits to form the holoenzyme at the peripheral region near the prolamellar body before transfer to the rudimentary pyrenoid.

Light-triggered organization of the stigma in dark-grown nondividing cells of Euglena gracilis.

Dark-grown cells of Euglena gracilis Klebs var. bacillaris Cori contain amorphous stigma material. When these cells are placed on resting medium for 3 days in darkness, the cells cease division; the organization of a normal stigma from the amorphous material requires continuous illumination for 72-96 hr. We have now found that if dark-grown cells are placed on resting medium for 8 days, a 40-min light pulse is sufficient to cause normal organization of the stigma in a subsequent 72-hr dark period. Thus stigma development is light-dependent at 3 days of resting but becomes light-triggered at 8 days. Other examples of light-triggered phenomena in Euglena are discussed and a model based on turnover of protein molecules repressing development that are ordinarily removed by exposure to light is presented; it is suggested that as the cells become more starved their ability to replace repressor molecules removed by light becomes limited and the system thereby becomes light-triggered rather than light-dependent.

Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells.

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called ;satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO(2)-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO(2)-fixation in photosynthesis.

Light-independent and dependent phases of proplastid development in Euglena gracilis W3BUL.

Cells of Euglena gracilis Klebs var. bacillaris Cori mutant W3BUL grown in darkness on Hutner's pH 3.5 medium without agitation accumulate wax ester. These cells have undeveloped proplastid remnants characteristic of this mutant. If these cells are transferred to an inorganic medium and bubbled with 2-3% CO2 in air, the wax disappears and the proplastid expands and develops in darkness to form prolamellar bodies and membrane vessicles within 96 h. No further development takes place in darkness, but if these cultures are illuminated at 96 h formation of prothylakoids is observed. Thus the wax ester accumulated during growth can be used subsequently to support proplastid development up to the prolamellar body stage, but the formation of prothylakoids is strictly light-dependent. Development in this system takes place at a slower rate than in cells grown with shaking and lacking wax which are transferred to resting medium. As previously shown, all of proplastid development requires light under these conditions. It is suggested that the oxygen-requiring utilization of wax in darkness can provide energy and metabolites for a part of proplastid development but the later steps in these cells, or the entire development in cells lacking wax is supported by paramylum degradation which is strictly light-dependent. However, a specific light reaction required for prothylakoid organization is not ruled out.

Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy.

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular 14C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr1BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.

Protein import into cyanelles and complex chloroplasts.

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, posttranslational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.

Fluorescence microscopic studies on mitochondria and mitochondrial nucleoids in a wild-type strain and respiratory mutants of Candida albicans.

A wild-type strain and two respiratory mutants of Candida albicans were examined for mitochondria and mitochondrial nucleoids (mt-nucleoids) using the fluorescent dyes, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4', 6-diamidino-2-phenylindole (DAPI). Rapidly growing cells of the wild type possessed one or a few giant branched mitochondria that were intensely stained with DASPMI. When a bud emerged, an end of the giant mitochondrion extended into the bud and the mitochondrion was divided and partitioned into mother and daughter cells by cytokinesis. Cell cycle-associated fragmentation or fusion of mitochondria were not demonstrated. The mutant KRD-8, that possesses cristate mitochondria but respires at a lower level, was shown to contain one or a few, less stainable giant mitochondria per cell. DASPMI failed to stain cells of the mutant KRD-19 which lacks cytochrome aa3 and cristate mitochondria. About eight and 10 mt-nucleoids were detected as discrete fluorescent spots in DAPI-stained cells of the wild type and KRD-8, respectively. KRD-19 cells also possessed mt-nucleoids, although the number of mt-nucleoids per cell seemed to be smaller than that of the wild type. In all the strains, mt-nucleoids existed discretely throughout the budding cycle, and the increase of their number per cell appeared to correlate with the cellular volume but not with nuclear division.

Isolation and characterization of mutants affected in lipid metabolism of Chlamydomonas reinhardtii.

Two mutants affected in lipid metabolism of Chlamydomonas reinhardtii were isolated by treating cells with ultraviolet light. Both mutants showed high chlorophyll fluorescent yields, as compared with parents, and were designated as hf-2 and hf-9 (for high fluorescence). hf-2 was shown to be defective in the synthesis of a chloroplast-specific lipid, sulfoquinovosyl diacylglycerol. hf-9 was shown to be defective in desaturation at the omega 6 position of fatty acids of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. The mutants exhibited alterations in photosynthetic activity and chloroplast ultrastructure.

Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes.

The reaction mechanism of bacteriophage T4 endonuclease V was investigated using modified oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. For the pyrimidine dimer glycosylase step, the formation of a covalent intermediate has been proposed. A fluorine atom was attached to the 2'-position of the 5'-component of the thymine dimer site, which could stabilize the covalent complex and prevent the ring opening of the sugar moiety. The strand cleavage of the 12 base pair substrate analog did not occur, although the glycosyl bond was cleaved by this enzyme. A covalent enzyme--substrate complex was separated by gel electrophoresis under denaturing conditions. It was shown that the enzyme molecules were completely converted to a stable complex in the reaction mixture. Two mechanisms have been proposed for the beta-elimination step. A 12-mer containing a phosphorothioate linkage between adjacent thymidines was prepared. The diastereomers were separated, and the absolute configurations were determined. After formation of the thymine dimer and 32P-labeling of the 5'-terminus, these oligonucleotides were annealed to the complementary 12-mer, and the reaction rates of the pyrimidine dimer glycosylase step and the overall reaction for each duplex were measured under the substrate-saturation conditions. The rate constants indicated that the chemical reaction at the beta-elimination step was rate-limiting. Since no difference was observed in the rate constants for the Rp- and Sp-phosphorothioate substrates, it is concluded that the beta-elimination reaction is catalyzed, not by the internucleotide phosphate, but by an amino acid residue of the enzyme.

Oxidative stress sensitivity and superoxide dismutase of a wild-type parent strain and a respiratory mutant of Candida albicans.

It is important to know responses of the pathogenic fungi to reactive oxygen species by which hosts protect themselves against fungal infection. In the present study, sensitivities to the superoxide radical (O2-) and superoxide dismutase (SOD) were compared between a wild-type parent strain and a respiration-deficient mutant of Candida albicans. When their survival was examined on an agar medium containing an intracellular O2- generator, paraquat (PQ), the parent strain was selectively killed by increasing the PQ concentration. In contrast, when cells of both strains were illuminated in a riboflavin solution, they exhibited similar sensitivity to O2- generated extracellularly by photo-reduced riboflavin. There were no large differences in sensitivity to hydrogen peroxide in the two strains. Thus, the high tolerance of the mutant to PQ was suggested to result from low intracellular O2- generation by PQ due to the respiratory deficiency. It is generally accepted that fungal cells contain manganese (Mn)-SOD in the mitochondria and copper and zinc (CuZn)-SOD in the cytoplasm. Cyanide-insensitive SOD activity (attributable to Mn-SOD) was dominant in the parent strain throughout growth phases, whereas cyanide-sensitive activity (attributable to CuZn-SOD) occurred in the mutant. The activity bands of Mn- and CuZn-SODs were clearly separated by electrophoresis of the cell extracts of both strains on non-denaturing polyacrylamide gels. The electrophoretic profiles obtained were consistent with the results of the activity assay. These results showed that the respiratory deficiency affected oxidative stress sensitivity and SOD in C. albicans.