Inorganello and invitro phosphorylation of chloroplast ribosomal proteins

Guitton, Carole; Dorne, Anne-Marie; Mache, Régis

doi:10.1016/0006-291x(84)90722-8

Cited by 15 publications

(6 citation statements)

References 12 publications

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“…Incorporation of 32P into the large subunit corresponds effectively to a phosphorylation since labelling is not removed by SDS/polyacrylamide gel electrophoresis and since large-subunit phosphorylation occurs on serine and threonine residues. We have observed another situation [17] with ribosomal proteins where phosphorylation occurs on a serine REFERENCES residue only, suggesting a different protein kinase activity. Foyer [12] has shown that a 55000-Mr polypeptide, comigrating with the large subunit, was labelled in organello but its phosphorylation could not be detected in isolated stroma even in the presence of NaF.…”

Section: Discussionmentioning

confidence: 81%

“…The trichloroacetic-acid-precipitated polypeptides were solubilized and separated by SDS-PAGE (12-20% gradient polyacrylamide gel) according to Laemmli [16]. Labelling was detected by autoradiography as previously described [17]. Markers of molecular mass are the following: phosphorylase b, 94000; bovine serum albumin, 67000; ovalbumin, 43 000; carbonic anhydrase, 30000; soybean trypsin inhibitor, 20000, a-lactalbumin, 14400.…”

Section: In Vitro Protein Synthesis and Electrophoresismentioning

confidence: 99%

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Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

Guitton

Mache

1987

Eur J Biochem

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A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 x g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-l,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-l,5-bisphosphate carboxylase/ oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.Many diverse physiological processes that occur in plants in response to light, growth regulators or to a stress are mediated via protein phosphorylation 11-31. Membranebound protein kinases, but also soluble ones, have been described in plant tissue 12, 4, 51.In chloroplasts, phosphorylation of proteins occurs mainly in thylakoids (on the light-harvesting chlorophyll-a/bbinding complex and on the N,N-dicyclohexylcarbodiimidebinding proteolipid) or in the chloroplast envelope [6 -lo]. To date polypeptide phosphorylated in the stroma of chloroplasts and soluble protein kinase have been poorly identified. Suss [l 11 has reported that light-exposed Vicia faba leaves incorporate 32P into a polypeptide comigrating with the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) after SDS/urea gel electrophoresis. But this author concluded that this polypeptide was not the large subunit because an immunoassay was negative. Recently another observation of phosphorylation of a polypeptide comigrating with the large subunit has been reported by Foyer [12] in spinach chloroplasts incubated with [32P]orthophosphate.In the present study we give evidence for the presence of a soluble chloroplast protein kinase able to phosphorylate stromal polypeptides two of them being the large subunit of Rubisco and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as demonstrated by immunological techniques. Some of the characteristics of large subunit phosphorylation are studied in vitro. MATERIALS AND METHODS Materials and reagentsSpinach plants (Spinaciu oleracea, var. Giant d'hiver) were grown on soil in a controlled room in the conditions already described [13] and were harvested after one month. Biochemicals were purchased from Sigma; RNasin, a RNase inhibitor, was from Genofit (Geneve); Percoll, Sephadex G-50, and protein-A-Sepharose CL-4B were from Pharmacia; Sorbitol was purified with mixed-bed resin (Bio-Rad AG 501-X8) and charcoal; [35S]methionine (43.3 TBq/mmol) ...

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Section: Discussionmentioning

confidence: 81%

Section: In Vitro Protein Synthesis and Electrophoresismentioning

confidence: 99%

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

Guitton

Mache

1987

Eur J Biochem

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“…4). The presence of a phosphorylated protein in plastid ribosomal 50 S subunit has been reported (54,55); the protein spot in those reports corresponds to PRPL19 (Figs. 1 and 8 (Table II), MS data would suggest no post-translational modification.…”

Section: Post-translational Processing: Plastid 50 S Rps Encoded In Tmentioning

confidence: 87%

The Plastid Ribosomal Proteins

Yamaguchi

Subramanian

2000

Journal of Biological Chemistry

199

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We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using twodimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs The plastid (chloroplast) ribosome is a plant-specific, organelle ribosome that produces proteins encoded by the plastid genome. Plastid ribosomes are responsible for the synthesis of huge amounts of biomass, since the large subunit of ribulose 1,5-bisphophate carboxylase/oxygenase (a most abundant protein in the biosphere) is synthesized in plastids. Plastid ribosomes are very similar to the eubacterial 70 S-type ribosome, in composition and general mode of function (1-4). The rRNAs and most of the characterized ribosomal proteins (RPs) 1 in plastid ribosomes also bear close resemblance to the corresponding components so far identified in cyanobacteria, a correlation supporting the importance of endosymbiotic theory in plastid evolution (5).The Escherichia coli ribosome, the most well studied of the eubacterial ribosomes (6), is composed of 21 RPs in the 30 S subunit and 33 RPs in the 50 S subunit. Two more possible E. coli RPs have been suggested: protein Y, the product of E. coli yfia gene (7), bearing a distant sequence homology to a chloroplast-specific RP (PSRP-1), and a protein designated S22 (8). Post-translational modifications are found in many E. coli RPs, although a modification in L16 (Arg 81 ) remains yet to be characterized (see "Results" for plastid L16). We have recently identified all the RPs in spinach plastid 30 S ribosomal subunit, including all its PSRPs and many post-translational modifications (83). The number of RPs in plastid 50 S subunits has so far only been estimated (ϳ35-39; reviewed in Ref.2) and has not been determined.Although the constituents of plastid translational machinery in general are similar to those of E. coli, the genes are distributed in two genome compartments: the plastid and the nucleus. The rRNA and tRNA genes are located in the plastid genome, whereas the genes for processing/modification enzymes, aminoacyl tRNA synthetases, and 60% of the RPs are located in the nuclear genome (1-4). The plastid translation system also differs from the eubacterial system in other significant ways, e.g. chloroplast mRNA is often edited (9); about 60% of chloroplast mRNAs lack canonical ribosome-binding sites found in E. coli mRNAs (10); mRNA levels in chloroplasts remain relatively unchanged through dark/light transitions, whereas protein

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“…Lightactivated protein phosphorylation in the chloroplast has been observed for both the thylakoid (Bennett, 1977;Lin et al, 1982;Mullet, 1983;Coughlan and Hind, 1986;Michel et al, 1988) and the stromal compartments (Guitton et al, 1984;Guitton and Mache, 1987;Sun et al, 1989). Lightactivated protein phosphorylation in the chloroplast has been observed for both the thylakoid (Bennett, 1977;Lin et al, 1982;Mullet, 1983;Coughlan and Hind, 1986;Michel et al, 1988) and the stromal compartments (Guitton et al, 1984;Guitton and Mache, 1987;Sun et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation.

1994

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Light‐regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5′‐untranslated region (5′‐UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5′‐UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark‐ and light‐grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA‐binding complex, that utilizes the beta‐phosphate of ADP to phosphorylate and inactivate psbA mRNA‐binding in vitro. The inactivation of mRNA‐binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark‐grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA‐binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.

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Inorganello and invitro phosphorylation of chloroplast ribosomal proteins

Cited by 15 publications

References 12 publications

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

The Plastid Ribosomal Proteins

ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation.

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