Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.     

Transport of peroxisomal proteins synthesized as large precursors in plants

Plant peroxisomes contain at least four proteins, namely, citrate synthase, malate dehydrogenase, long-chain acyl-CoA oxidase, and 3-ketoacyl-CoA thiolase, which are synthesized as large precursors with an N-terminal cleavable presequence. Each presequence has a conserved domain (R[I/L./Q]-X₅-HL) that is homologous to peroxisomal targeting signal 2 from mammals and yeasts. In addition, a cysteine residue is found at the C-terminal ends of the presequences, whose function has not yet been described. The authors analyzed the function of the presequences and the conserved amino acids using transgenic Arabidopsis plants, which accumulate β-glucuronidase carrying the presequence of the peroxisomal proteins from plants. Immunological and immunocytochemical studies on the transgenic plants showed that a conserved sequence in the extrapeptides is essential for targeting to peroxisomes, and a cysteine residue at the cleavage site is involved in the processing of the presequence. These results suggest that the presequences of the peroxisomal proteins function as targeting signals, and are necessary for the recognition of the processing.     

Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.     

Chloroplast elongation factors are synthesized in the chloroplast.

The elongation factor G (EF-Gchl) and elongation factor Tu (EF-Tuchl) present in spinach chloroplasts become labelled when isolated chloroplasts are incubated in the light with radioactive methionine. EF-Gchl and EF-Tuchl account for approximately 0.04% and 0.2% respectively of the total radioactivity incorporated by isolated organelles.     

Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga.     

Organelle-bound malate dehydrogenase isoenzymes are synthesized as higher molecular weight precursors

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).     

衣藻叶绿体遗传转化的研究

叶绿体遗传转人是近几年发展起来的新领域。本文主要介绍了叶绿体遗传转化的特点、基本原理和衣藻叶绿体遗传转化的方法与技术;     

Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli

Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii. Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported. In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C. reinhardtii. Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight. Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E. coli large subunit protein. None of the antisera cross-reacted with any E. coli small subunit proteins. On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E. coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis. Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C. reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain.      

mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.     

Engineering the Chloroplast Encoded Proteins of Chlamydomonas

Over a decade ago (1988), John Boynton and colleagues successfully transformed the chloroplast genome of chlamydomonas for the first time by complementation of a chloroplast deletion mutant. Since the first demonstration of chloroplast transformation the function and structure of many chloroplast encoded subunits of the photosynthetic apparatus has been characterized by site-directed mutagenesis. With the completion of the sequencing of the Chlamydomonas chloroplast genome the genetic tools are now in hand to characterize structure-function relationships for each of the chloroplast-encoded proteins of the photosynthetic apparatus.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.