Tetsuaki Osafune

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.

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, post-translational 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.

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.

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6‐diamidino‐2‐phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp‐nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread‐like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread‐like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI‐stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.

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.

SUBCELLULAR LOCALIZATION OF IRON AND MANGANESE SUPEROXIDE DISMUTASE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

Subcellular localization of superoxide dismutase (SOD) isozymes in Chlamydomonas reinhardtii Dangeard was investigated. From both biochemical and immunological studies, Fe‐ containing superoxide dismutase (Fe SOD) was localized in the chloroplast, and the major Mn‐containing SOD (Mn SOD) was localized in the mitochondria. We isolated cDNA clones, Sod1, and Sod2, which encode Mn SOD and Fe SOD, respectively. Genomic DNA hybridization with these SOD clones demonstrated that there are two additional sequences closely related to Sod1. The transcriptional levels of both genes were compared in light‐ and dark‐grown cells. Mn SOD was higher in heterotrophically grown cells, whereas the accumulation of Fe SOD transcripts was higher under phototrophic condition.

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra‐serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper‐binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Accumulation of LHCP II apoprotein in wax-rich cells of Euglena in low light or in the presence of streptomycin

Abstract Cells of Euglena gracilis var. bacillaris or Z strain grown under the usual conditions (“nonwax cells”) and exposed to low-intensity light at the developmental threshold (3–7 ft-c) fail to accumulate LHCP II apoprotein. However, cells grown in a medium rich in hexose without shaking accumulate wax; these wax-rich cells, after subsequent aeration in darkness on an inorganic medium for 6 days, accumulate LHCP II apoprotein on exposure to low-intensity light. Immunoelectron microscopy of these cells using anti-LHCP II antibody and protein A—gold shows LHCP II apoprotein first in the compartmentalized osmiophilic body (COS) and Golgi apparatus followed by the thylakoids of the plastid, as previously seen in nonwax cells at normal light intensities for chloroplast development. With time in the light the aerated wax-rich cells at low light intensity form curly thylakoids which react with the LHCP II antibody; exposure of these cells to high light intensity (500 ft-c) causes the curly thylakoids to assume the more normal straight configuration and these retain the reaction with LHCP II antibody. Aerated dark-grown wax-rich cells exposed to normal light intensities for chloroplast development (150 ft-c) in the presence of 0.1% streptomycin have plastids in which the disappearance of the prolamellar body (PLB) is inhibited; a paracrystalline body is formed in close proximity to the PLB which shows an immunoreaction with LHCP II antibody, but the immunoreaction is absent from the thylakoids. Thus, conditions in the aerated wax-rich cells allow an unusual accumulation of LHCP 11 apoprotein at low light intensities and streptomycin blocks the distribution of this apoprotein to the thylakoids in these cells at normal intensities.

The occurrence of non-specific lipid transfer proteins in developing castor bean fruits

Abstract The occurrence of non-specific lipid transfer proteins (nsLTPs) in developing castor bean fruits was examined by immunoblot quantitation and immunogold electron microscopy. The levels of nsLTPs increased with fruit development, being higher in the pericarp than the seed. The distribution of nsLTPs was localized throughout the development in the peripheral tissues, the outer layer of the endosperm in the seed and the epicarp in the pericarp. nsLTPs in the endosperm were localized only in the dense vesicle in the cytoplasm at 14-days after flowering (14-DAF). At 28-DAF the dense vesicles containing nsLTPs were assembled to the central vacuole to form an aggregate, which in turn disappeared at 35-DAF. On the other hand, nsLTPs were scattered in the cell walls of sclereid surrounding the seed and in the cell walls of trichome and sclerenchyma in the pericarp. These results suggest that gene-expressed nsLTPs are transported by two routes; one is to the vacuole by the dense vesicles in the endosperm and the other is to the cell wall and around there in the pericarp, probably by Golgi route.

Serial Section Immunoelectron Microscopy of Algal Cells

Electron microscopy when combined with immunogold labeling provides a 2D image of intracellular protein distribution. Cells are however 3D structures. We describe a method of serial section immunogold electron microscopy that allows a 3D cellular image to be reconstructed from a series of electron micrographs. Cells are fixed to preserve cellular ultrastructure and they are embedded in plastic allowing ultrathin sections to be obtained. The ribbon of ultrathin serial sections produced as the microtome sequentially cuts through the sample is labeled with a monospecific antibody to the protein of interest and then with protein-A gold making the antigen-antibody complex visible in the electron microscope. A common field of view from each serial section is photographed in the electron microscope. Using image analysis software, each digitized micrograph is sequentially aligned; immunolabel and cellular structures of interest are traced onto each micrograph; the micrographs are stacked; and the structures of interest are rendered as solid surfaces producing a 3D image of protein distribution within the cell.