Processing of a Chloroplast‐Translated Membrane Protein <i>in vivo</i>

Reisfeld, Avi; Mattoo, Autar K.; Edelman, Marvin

doi:10.1111/j.1432-1033.1982.tb05914.x

Cited by 93 publications

(51 citation statements)

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“…Several investigators have reported on the synthesis in chloroplasts of a 32,000 dalton membrane polypeptide during development of plastids (3,14,37,44,55). This protein is syr~4;hesized as a larger precursor of species dependent size and is processed to its natural size during incorporation into the photosynthetic membranes probably as a component of photosystem II.…”

Section: Light Regulated Rna Synthesismentioning

confidence: 99%

The barley chloroplast genome: Physical structure and transcriptional activity in vivo

Poulsen

1983

Carlsberg Res. Commun.

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Keywords: Chloroplast DNA, restriction endonucleases, the large subunit of ribulose bisphosphate carboxylase, chloroplast RNA, formaldehyde agarose gels, Northern blotting, filter hybridization, light induced transcription.DNA has been isolated from barley chloroplasts and analyzed with restriction endonucleases PstI, Sail, PvulI and HindIII. Fragments obtained by digestion with PstI and HindIII have been inserted in the bacterial transformation vectors pBR325 and pBR322 and amplified in Escherichia coli. The inserts of the recombinant plasmids have been mapped with the same restriction enzymes as the intact chloroplast DNA yielding a physical map for the barley chloroplast genome. It is a circular molecule about 133,000 basepairs in size, which is equivalent to the chloroplast genomes of other gramineae such as wheat and maize. Like these it has an inverted repeat, of about 20,900 basepairs, containing the genes for the ribosomal RNAs. There are many similarities of the barley, wheat and maize genomes with respect to recognition sites for the enzymes PstI, SalI and PvuII. Heterologous hybridization with a probe containing parts of the genes for the large subunit of ribulose bisphosphate carboxylase and for the dicistronic mRNA for two ATPsynthetase CF~ subunits, reveal that the position and organization of these genes in the barley chloroplast DNA are the same as found for maize and wheat.Selected chloroplast DNA fragments isolated from the recombinant plasmids and covering about 80% of the genome have been used for hybridization to RNA purified from plastids of dark grown and 8 hours illuminated seedlings. The RNA was electrophoretically separated in denaturing agarose gels and subsequently Northern blotted onto DBM-paper. The DNA fragments were nick translated and hybridized to the filterbound RNA. A total of 70 transcripts ranging in size from 350 nucleotides to more than 6,000 were identified. Of these 11 were synthesized only after illumination of the seedlings. The transcripts encoding the large subunit of ribulose bisphosphate carboxylase and the ATP synthetase CFt subunits [3 and e have been tentatively identified. About 30 transcripts are larger than 3,000 nucleotides, indicating either the existence of a large number of polycistronic mRNAs or various forms of precursor RNAs.Abbreviations: bp = basepairs; DBM-= diazobenzyloxymethyl-; DCCD = dicyclohexylcarbodiimide; DCMU = dichlorophenyl-dimethyl-urea; kb = kilobases; kbp = ldlobasepairs; LS = large subunit; NBM-= nitrobenzyloxymethyl-; NBPC = nitrobenzyloxy-methylpyridinium chloride;pHvC = recombinant plasmids carrying Hordeum vulgare chloroplast DNA fragments; RuBPCase = ribulose bisphosphate carboxylase. Genes: rbcL = the gene for RuBPCase LS; atpA = the gene for ATP synthetase CFt subunit ct; atpB = the gene for ATP synthetase CF~ subunit [3; atpE = the gene for ATP synthetase CF 1 subunit e; atpH = the gene for ATP synthetase CFo subunit III; psbA = gene for a 32,000 dalton photosystem II protein; rrs, rrl and rrf= 16S, 23S and 5S rRNA genes; trn ...

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Section: Light Regulated Rna Synthesismentioning

confidence: 99%

The barley chloroplast genome: Physical structure and transcriptional activity in vivo

Poulsen

1983

Carlsberg Res. Commun.

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“…PCC 6803, there is no evidence for a "33.5-kD" D1 precursor as observed in plants (Reisfeld et al, 1982;Marder et al, 1984;Minami and Watanabe, 1985;Inagaki et al, 1989). However, this may be due to a decreased life time of the precursor compared with the mature protein in cyanobacteria and does not imply that there would be a fundamental difference between D1s from plants and cyanobacteria in this respect.…”

Section: Steady-state Levels Of Photosystem II Thylakoid Proteins Andmentioning

confidence: 72%

Transcript Levels and Synthesis of Photosystem II Components in Cyanobacterial Mutants with Inactivated Photosystem II Genes.

Vermaas

1990

Plant Cell

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After interruption or deletion of the photosystem II genes psb8, psbC, and psbD in the cyanobacterium Synechocystis sp. PCC 6803, thylakoids from such mutants were found to be depleted in a number of photosystem II proteins in addition to those for which the gene(s) had been inactivated. Transcript levels of photosystem II genes were measured and protein pulse-labeling was carried out to determine the reason for this effect. Transcripts of all photosystem II genes except the inactivated one(s) were found to be present in the various mutants. In certain cases, inactivation of one photosystem II gene led to overexpression of another. Protein pulse-labeling experiments using 35S-methionine, in which not only the rapidly turning over D1 protein but also D2, CP43, and CP47 appear to be preferentially labeled, showed that the mutants studied synthesize the D1 protein as well as other photosystem II proteins whose genes were not inactivated. The fact that, in the various mutants, photosystem II proteins for which the gene is not inactivated are synthesized but do not accumulate in the thylakoid indicates that the psb8, psbC, and psbD gene products are all required for a stable assembly of the photosystem II complex.

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“…First, it is known that the Qb is an integral membrane protein and cannot be removed from the membrane by high salt concentrations (23). Second, tryptic digestion of the membrane form of the Qb yields two well defined polypeptides which remain with the membrane (1, 18).…”

Section: Resultsmentioning

confidence: 99%

In Vitro Synthesis, Assembly and Function of a Photosynthetic Membrane Protein

Michaels

Herrin

1989

Plant Physiol.

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Cell-free translation of Chiamydomonas reinhardtli RNA in the presence of photosynthetic membranes resulted in association of the herbicide binding (0b) protein with membranes. Incubation of recovered membranes with high salt did not extract the polypeptide from membranes. Tryptic digestion of in vivo labeled membranes or membranes recovered from in vitro translation mixtures showed that Qb had similar orientation. In vitro translation in the presence of chloroplast membranes from cells exposed to high light intensity restored the membrane associated kinase activity lost by photoinhibition. Thus, in vitro synthesis resulted in functional integration of the Qb protein within the photosynthetic membrane.There is increasing evidence that at least some chloroplast thylakoid membrane proteins are synthesized by polyribosomes bound to photosynthetic membranes (5, 17). We and others have shown that thylakoid-bound ribosomes of higher plants and algae produce a number of photosynthetic membrane proteins (3, 6-8, 10, 11, 13-15 Total cellular and poly(A) lacking RNA was prepared as described (9). Translation of RNA in the presence or absence ofthylakoids was accomplished in a reticulocyte extract (New England Nuclear) for 1 h as specified by the supplier. Cycloheximide (10 ,g/mL) was added to all in vitro translation reactions and thylakoids were added to those reactions not already containing them. Incubation was continued for an additional 0.5 h after which thylakoids were recovered from all samples, washed by centrifugation with 25 mm Tricine (pH 7.5), and resuspended in electrophoresis sample buffer. Washed thylakoids, recovered from translation mixtures, were also resuspended in buffer with and without 1 M LiCl, incubated at 0C for 0.5 h, washed by centrifugation with 25 mM Tricine (pH 7.5), and resuspended in electrophoresis sample buffer. Trypsin treatment of recovered thylakoids was according to published methods (16,18).Photoinhibition and PAGE analysis of the light dependent membrane kinase activity using [a-32P]ATP as substrate was performed as described (7,21). Thylakoids from photoinhibited cells were either used immediately after isolation or stored at -80C (19). Stored thylakoids used within 1 week of isolation did not show light dependent phosporylation. However, the thylakoids did exhibit light independent phosphorylation in the presence of duroquinol (data not shown) (24). Addition of synthetic electron donors to PSII (e.g. diphenylcarbazide) did not increase light dependent phosphorylation in control or reconstituted reactions. RESULTS AND DISCUSSIONPhotosynthetic membranes without bound ribosomes were isolated from C. reinhardtii grown photoautotrophically (1 1). The membranes did not stimulate in vitro translation without addition of exogenous RNA. This is consistent with our previous results that showed antibiotic stabilization of polysomes on thylakoids (17). Without IN VITRO RECONSTITUTION OF PHOTOSYNTHETIC MEMBRANES[35S]methionine, washed, and analyzed by electrophoresis and fluorograph...

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Processing of a Chloroplast‐Translated Membrane Protein in vivo

Cited by 93 publications

References 23 publications

The barley chloroplast genome: Physical structure and transcriptional activity in vivo

The barley chloroplast genome: Physical structure and transcriptional activity in vivo

Transcript Levels and Synthesis of Photosystem II Components in Cyanobacterial Mutants with Inactivated Photosystem II Genes.

In Vitro Synthesis, Assembly and Function of a Photosynthetic Membrane Protein

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