CHLOROPLAST RIBOSOME BIOGENESIS IN <i>CHLAMYDOMONAS  </i>

Harris, Elizabeth H.; Boynton, John E.; Gillham, Nicholas W.

doi:10.1083/jcb.63.1.160

Cited by 67 publications

(40 citation statements)

References 42 publications

(35 reference statements)

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“…Third, the heteroplasmic orf722 mutant has a growth phenotype similar to that of previously characterized mutants with decreased chloroplast ribosome functions. These mutants grow at or near wild-type rate under heterotrophic or mixotrophic conditions, but are unable to grow under phototrophic conditions [20,22]. This is not surprising, considering that chloroplast ribosomes synthe- size a large number of chloroplast-encoded proteins that are required in photosynthesis (e.g.…”

Section: Discussionmentioning

confidence: 96%

“…Second, Orff 12 is functionally essential for cell growth, just as expected if it is a ribosomal protein. In Chlamydomonas, chloroplast ribosome function is essential for cell viability, although it can be reduced to a minimal level required for cell growth under heterotrophic or mixotrophic conditions [5,19,20]. This is presumably due to a necessity to make one or more chloroplast-encoded proteins that have essential cellular functions, as has been suggested for plants [21].…”

Section: Discussionmentioning

confidence: 99%

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The unusual rps3‐like orf712 is functionally essential and structurally conserved in Chlamydomonas

Liu

Huang

1993

FEBS Letters

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The Chlumydomonas reinhardtii chloroplast orf712 is a previously described open reading frame that lacks a detectable transcript but potentially encodes a polypeptide with sequence similarities to ribosomal protein Rps3 only at its N-and C-termini. Here we report that orj712 is an essential gene, as demonstrated through gene disruption by particle gun-mediated chloroplast transformation. We also show that an orf712 is present and structurally conserved in all of the two or three major Chlumydomonus lineages. Our results suggest that orpI is an unusual rpd gene that contains a large translated intervening sequence.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

The unusual rps3‐like orf712 is functionally essential and structurally conserved in Chlamydomonas

Liu

Huang

1993

FEBS Letters

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Section: Resultsmentioning

confidence: 99%

“…The mutations are unlinked (3), exhibit Mendelian inheritance (3), and affect distinct steps of chloroplast ribosome assembly (14). Whereas the ac2O mutation apparently disrupts the formation of both 41 S and 54S ribosomal subunits, crl acts on the 41S subunit alone (14). The primary lesion in these cells, markedly reduced levels of chloroplast ribosomes (14,24), leads to various functional and structural deficiencies whose cause, in some cases, has been traced to the lack of chloroplast-synthesized gene products.…”

Section: Resultsmentioning

confidence: 99%

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

Mishkind

Schmidt²

1983

Plant Physiol.

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The coordinated synthesis of the subunits of ribulose 1,5-bisphosphate carboxylase was examined by analysis ofthe ch}oroplast ribosome-deficdeat mutant of Chiamydomonas re ardtii, ac2Ocrl. The absence of the chloroplast-synthesized large subunit of this enzyme from cells of this strain is a direct consequence of the lack of chloroplast nbosomes. In contrast, the absence of the cytoplasmicafly-synthesized small subunit of ribuose 1,5-bisphosphate carboxylase from this mutant is not understood. To discern the cause of this absence, we have compared results of in vivo radioactive labeling experiments with those of ceDl-free translations of RNA from ac20crl. Protein products from these experiments were identified by oneand two-dimensional electrophoretic analyses. Neither subunit, revealed either as a stained band or by fluorography ofproteins radioactively labeled in vim for 2 hours, was detected in ac2Ocrl. Cell-free translation of polyadenylated RNA obtained from ac20crl, however, revealed wild-type levels of mRNA for the precursor to the small subunit. This messenger was found to be associated with subribosomal RNP and polysomes. We conclude that the absence of the small subunit of ribulose 1,5-bisphosphate carboxylase from ac2Ocrl is the result of a translational or posttranslational event.in ac20crl at wild-type levels during heterotrophic growth (24), free pools of the cytoplasmically synthesized S of RuBPCase are not detected in these cells (9). Thus, there appears to be a specific interaction between the chloroplast and cytoplasm that serves to regulate the accumulation of S. The experiments in this paper were designed to identify the site(s) at which control of S synthesis is exerted. We demonstrate by in vivo labeling experiments analyzed by two-dimensional pH gradient gel electrophoresis that pools ofpS as well as pools of S itself are not present in detectable levels in ac2Ocrl. We also find that heterotrophically grown cells of ac20crl contain wild-type levels of mRNA for this subunit and that some of this messenger is present on polysomes. Therefore, 0

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“…Screening for mutants with enhanced chlorophyll fluorescence (5) (6,7). The shortcomings of the arsenate procedure include an unknown mode of action and the prevalence of arsenate permeability mutants among the recovered strains (8). Because mutants that are blocked in photosynthetic CO2 fixation are also isolated (7,8), arsenate resistance is not a property restricted to those with impaired photosynthetic electron transport.…”

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confidence: 99%

A rapid procedure for selective enrichment of photosynthetic electron transport mutants.

Schmidt¹,

Matlin²,

Chua³

1977

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

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Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) is shown to be effective for the selective enrichment of mutants of Chlamydomonas reinhardtii that possess impaired photosynthetic electron transport. More than 99.91% of -type cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under identical conditions.The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-dependent oxygen utake mediated by metronidazole is shown to require active photosystem I in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid membrane fractions enriched in photosystem I activity. We propose that the toxicity of metronidazole is due to the formation of lethal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metronidazole reduced by one electron. Preparation of Thylakoid Membrane Fractions. Thylakoid membranes were purified as previously described (S). For assays of photosynthetic electron transport activities, the thylakoids were suspended in 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes)-KOH (pH 7.5).Preparation of PS I Particles. Preparations of thylakoid membrane particles enriched in PS I activities were obtained by digitonin treatment of crude membranes (10). Wild-type cells (8 X 108) were harvested by centrifugation at 2500 X g and washed with 10 mM potassium phosphate (pH 7.0)/20 mM KCI/2.5 mM MgCl2. Washed cells were resuspended in 40 ml of the same buffer and were disrupted at 00 by sonication for 1 min with a Heat-System Ultrasonics sonifier. Pellets obtained by centrifugation at 48,000 X gma. for 15 min were resuspended in 15 ml of the buffer and homogenized with a motor-driven Teflon pestle. The thylakoids (300 ,g of chlorophyll per ml) were treated with 0.5% (wt/vol) RESULTSMetronidazole (2-methyl-5-nitroimidazole-1-ethanol) is widely used clinically for the treatment of infections of anaerobic bacteria and protozoa (11). It is a nitroheterocyclic compound possessing a low redox potential (-325 mV at pH 6.9) (12). The toxicity of metronidazole is thought to depend upon reduction 610

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CHLOROPLAST RIBOSOME BIOGENESIS IN CHLAMYDOMONAS

Abstract: Chloroplast protein synthesis in Chlarnydomonas reinhardtii is dispensable when cells are provided acetate as a carbon source. Mutants defective in synthesis, assembly, or function of chloroplast ribosomes are therefore conditionally viable.

Cited by 67 publications

References 42 publications

The unusual rps3‐like orf712 is functionally essential and structurally conserved in Chlamydomonas

The unusual rps3‐like orf712 is functionally essential and structurally conserved in Chlamydomonas

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

A rapid procedure for selective enrichment of photosynthetic electron transport mutants.

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