Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast.

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.     

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.      

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.     

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts

Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.     

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit

Summary Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.     

Biosynthetic pathways of two polypeptide subunits of the light- harvesting chlorophyll a/b protein complex

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post- translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.     

Transport of proteins into chloroplasts. Binding of nuclear-coded chloroplast proteins to the chloroplast envelope

A system has been constructed in vitro for the binding of cytoplasmically synthesized chloroplast proteins to the chloroplast envelope which precedes the uptake into the organelle in vivo. Isolated chloroplast envelopes from young pea or spinach are capable of binding the majority of proteins obtained by translation of poly(A)-containing RNA from greening plants in vitro. Among the bound proteins the precursors to the light-harvesting chlorophyll a/b apoprotein and the small subunit of ribulose-1,5-bisphosphate carboxylase are prominent. Binding is an intrinsic property of the envelope membrane and does not require energy in the form of ATP. Bound proteins remain on the surface of the envelope vesicles and can be digested by protease. Binding is complete within minutes, shows a high affinity of the reactants, and is non-ionic in nature. Protein binding is specific for translation products of poly(A)-containing RNA from greening plants. Precursors to chloroplast protein are bound preferentially as compared to the mature proteins. The specificity is further demonstrated by the low binding of proteins obtained by run-off translation of polysomes. Binding of radioactive labeled proteins is subject to competition by excess unlabeled homologous proteins. Once bound, the proteins are withdrawn from competition indicating a high binding stability. All the properties found for binding of proteins to isolated envelopes are consistent with the concept of the so-called envelope carrier hypothesis.     

Isolation of nuclear encoded plastid ribosomal protein cDNAs

Summary A pea leaf cDNA library was constructed in the expression vector gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500–5,500 daltons smaller than their precursors.     

Optimal conditions for post-translational uptake of proteins by isolated chloroplasts. In vitro synthesis and transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase

Many polypeptides translated in the cytosol enter the chloroplast where they assemble into macromolecular complexes. The transport of these polypeptides into the plastid can be examined in vitro by mixing isolated chloroplasts with pea poly(A) RNA translation products. Following optimization of both translation in the wheat germ system and the conditions during in vitro uptake, we observe the post-translational transport of over 100 polypeptides; many remain in the soluble phase of the organelle while others integrate into the thylakoid membranes. Most products transported in vitro co-migrate with in vivo products on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, with the improved conditions, we demonstrate the transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase into isolated plastids. While we have not been able to detect any cell-free translation product that is immunologically related to fructose-1,6-bisphosphatase, both plastocyanin and ferredoxin-NADP+ oxidoreductase are synthesized as precursors in vitro. These precursors are imported into the organelle where they are processed to the size of their mature counterparts. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the precursor to plastocyanin is 15,000 larger than the mature product and the precursor to ferredoxin-NADP+ oxidoreductase is 8,000 larger than the mature product.     

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

Chloroplast ribosomal protein L-18 in Chlamydomonas reinhardtii is processed during ribosome assembly

Summary Chloroplast ribosomal protein L-18 is made in the cytoplasm as a precursor, imported into the chloroplast, and processed to the mature form in two steps. We report here that the intermediate produced following the first processing step associates specifically with a ribosomal complex migrating with the chloroplast ribosome large subunit peak in sucrose gradients, and is then processed into mature L-18. This processing event is slowed down in mutant cells deficient in synthesis of non-ribosomal proteins in the chloroplast. Thus the second processing step of L-18 occurs during ribosome assembly, depends on one or more nonribosomal proteins made in the chloroplast, and may be required for the maturation of the 50 S ribosome subunit. The mature L-18 protein shows extensive sequence homology at its amino-terminus to Escherichia coli ribosomal protein L27, which is located at the interface, between 30 S and 50 S subunits and is involved in the formation of the peptidyl-tRNA binding site.     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.     

Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.     

Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Chlamydomonas reinhardi

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi. Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.     

Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism

A proteomic analysis of Chlamydomonas reinhardtii 70S ribosomes identified two proteins, RAP38 and RAP41, which associate in stoichiometric amounts with intact ribosomes. In this work we show results that suggest the Arabidopsis thaliana homologs, CSP41b and CSP41a, participate in ribosomal RNA metabolism. Csp41a-1 and csp41b-1 single mutants show little phenotype, while the loss of both proteins is lethal. Plants homozygous for the csp41b-1 mutation and heterozygous for the csp41a-1 mutation (csp41b-1/csp41a-1*) fail to accumulate CSP41b and show a marked reduction in the levels of CSP41a. These mutants have reduced chlorophyll content, grow slower and over-accumulate 23S precursor rRNAs compared to their wild-type (WT) siblings, whereas other rRNAs or mRNAs are unaffected. Chloroplast polysome assembly is reduced in csp41b-1/csp41a-1* mutants, which also contain increased amounts of pre-ribosomal particles compared to mature 70S ribosomes. Our results also indicate that CSP41b associates with pre-ribosomal particles in vivo. In vitro, the pattern of 23S precursors and mature rRNAs is altered upon incubation with recombinant CSP41a and CSP41b. Taken together, these results suggest that CSP41a and CSP41b have a role in chloroplast ribosomal RNA metabolism, most likely acting in the final steps of 23S rRNA maturation.