Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in <i>Chlamydomonas reinhardtii</i>

Mishkind, Michael; Schmidt, Gregory W.

doi:10.1104/pp.72.3.847

Cited by 48 publications

(21 citation statements)

References 12 publications

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“…Therefore, the in vivo transit of pS from cytoplasm through the chloroplast envelope and its maturation occurs in less than 5 min. (lanes 1-10) and then chased with unlabeled Na2SO4 for 60 min (lanes [11][12][13][14][15][16][17][18][19][20] The time course for chloramphenicol inhibition of chloroplast protein synthesis and onset of degradation of small subunit is shown in Fig. 4.…”

Section: Resultsmentioning

confidence: 99%

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts

Schmidt

Mishkind

1983

Proc. Natl. Acad. Sci. U.S.A.

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226

118

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We have detected a proteolytic mechanism in chloroplasts that selectively and rapidly degrades the imported small subunit of ribulose 1,5-bisphosphate carboxylase when pools of the chloroplast-synthesized large subunit are depleted. This degradation system is constitutively present and appears to be responsible for precise stoichiometric accumulation of the two subunits of the enzyme. We believe similar proteolytic mechanisms participate in regulating the accumulation of other photosynthetic proteins during chloroplast biogenesis.Ribulose 1,5-bisphosphate carboxylase/oxygenase [RbuP2Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the major protein in C3 plants, is localized in the soluble fraction of chloroplasts, where it catalyzes the initial steps in CO2 reduction (1, 2). The RbuP2Case holoenzyme consists of eight copies of each of the 52,000-to 56,000-dalton large subunit and the 12,000-to 15,000-dalton small subunit. Much is known concerning the mode of synthesis of the large subunit within chloroplasts and of the nuclear-encoded small subunit in the cytoplasm. However, many aspects of the regulation of synthesis and the pathway for subunit assembly remain to be elucidated. In particular, the mechanism(s) by which stoichiometric amounts of large and small subunits are produced during chloroplast biogenesis have not been established. In this paper, we provide evidence that, when the small subunit is produced in excess of the large, it is selectively and rapidly degraded by a proteolytic activity that is constitutively present in chloroplasts. MATERIALS 43 Ci/mg at 100% isotopic enrichment; 1 Ci = 37 GBq; ICN Chemical and Radioisotope Division) was added to a final concentration of 1 mCi/ml. The chase periods were begun on addition of a 1/10th vol of 100 mM Na2SO4. At intervals, 100-,ul aliquots were transferred to 900 ul of 100% acetone at 0°C to terminate protein synthesis and degradation. Electrophoresis. Acetone precipitates were dissolved in 2% NaDodSO4/60 mM Tris HCI, pH 8.6/60 mM dithiothreitol/5 mM E-aminocaproic acid/i mM benzamidine/15% (wt/vol) sucrose, and 5-1Al aliquots were removed for radioactivity determination (4). Equal amounts of labeled protein were subjected to electrophoresis in 10-20% (wt/vol) gradient polyacrylamide gels prepared in the buffer system of Laemmli (5). For two-dimensional electrophoresis, the precipitates were dissolved in 2% Ampholines (pH 3.5-10; LKB)/2% Nonidet P-40 (Particle Data Laboratories)/10 mM dithiothreitol/9.5 M urea/10 mM methylamine and subjected to nonequilibrium pH gradient electrophoresis for 1,600 V-hr as described (6), except that the formulation of ampholytes for a pH range of 3.5 to 9.5 (7) was used. Electrophoresis in the second dimension was carried out in the NaDodSO4/polyacrylamide gradient gels described above. Gels were treated for fluorography (8) before exposure to Kodak X-Omat AR film at -70°C. RESULTSTo study the in vivo events involved in the synthesis of RbuP2Case, we have used Chlamydomonas in pulse-chase ...

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Section: Resultsmentioning

confidence: 99%

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts

Schmidt

Mishkind

1983

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

226

118

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“…Proteins were synthesized in vitro using the wheat germ cell-free system and 35S-methionine (DuPont NEN) (Mishkind et al, 1987).…”

Section: In Vivo and In Vitro Labeling Of Proteinsmentioning

confidence: 99%

“…Samples were resuspended in gel sample buffer (60 mM Tris-HCI, pH 8.6, 60 mM DTT, 5 mM E-amino-n-caproic-acid, 1 mM benzamidine, 2% LDS, 12.5% sucrose) and resolved by PAGE, as described by Mishkind and Schmidt (1983). Proteins were detected with Coomassie Brilliant Blue R 250.…”

Section: Lds-page and Protein Blottingmentioning

confidence: 99%

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly.

Plumley

Schmidt

1995

Plant Cell

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The biogenetic interdependence of light-harvesting chlorophyll (Chl) alb proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP 11) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesidassembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.

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“…In pea light increases the transcription of the small and large subunit genes (29). In a Chlamydomonas mutant deficient in chloroplast ribosomes mRNA for the small subunit is produced at normal levels but not translated (19).…”

Section: Introductionmentioning

confidence: 99%

Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

Poulsen

1984

Carlsberg Res. Commun.

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The region of the barley chloroplast genome encoding the 5' third of the rbeL gene has been subcloned in M 13 vectors and sequenced by the dideoxy chain termination method. Some of the recombinant M 13 clones have been used as a template for the synthesis of highly radioactive single stranded DNA probes. These are specifically hybridizing to short regions at the 5' end of the two transcripts, which were previously found to be encoded by the rbcL region in barley cpDNA. The two transcripts differ in size by more than 200 nucleotides, are encoded by the same strand and contain the full coding capacity for the large subunit of ribulose bisphosphate carboxylase-oxygenase. The short transcript is predominant in dark grown seedlings. Of two probes employed in this study one hybridized only to the long and the other to both rbcL mRNA species. Thereby it was possible to quantitate the rbeL m RNAs by hybridization with the probes and subsequent S l-nuclease digestion of the single stranded molecules. I found this mRNA to be aproximately 1.38% of total plastid RNA (excluding the small rRNAs and the tRNAs) in dark grown seedlings and 2. 20% in greened seedlings. The two probes were also used in primer extension experiments using the two transcripts as template. After hybridizing the probe to the mRNA reverse transcription was performed in the presence of cold deoxynucleotides. The reverse transcribed strands were separated on sequencing gels. The probe which acted as a specific primer for the short transcript was extended by 29 nucleotides, and the probe specific for the longer transcript was extended by 91 nucleotides. The end point of the latter transcript was only found with template RNA from plastids of greened barley seedlings. Thus it was shown that the 5" ends of the two transcripts begin at positions -58 and -316 from the ATG triplet of the rbcL reading frame. A comparison of the sequences upstream from the ATG codon of the rbeL gene of barley with those of maize, spinach and tobacco revealed completely conserved prokaryote type transcription promotors in front of the 5' end of the long transcript, while no such promotors are evident close to the 5' end point of the short transcript. The two monoeot species differ from the dicot species by an insertion of about 140 nucleotides in the mRNA leader sequence.Abbreviations: atpB = the gene for ATP synthetase CF, subunit ~; bp(s) = basepair(s); cpDNA = chloroplast DNA; dsDNA = double stranded DNA; kb(s) = kilobase(s); kbp(s) = kilobasepair(s); LS = large subunit; psbA = gene for a 32,000 dalton photosystem II protein; rbeL = gene for the RuBPCase-Oase LS; RF = replicative form; RuBPCase-Oase = ribulose bisphosphate carboxylase-oxygenase; ssDNA = single stranded DNA.Springer-Verlag

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Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in Chlamydomonas reinhardtii

Cited by 48 publications

References 12 publications

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly.

Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

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