MEMBRANE-ASSOCIATED RIBOSOMES IN MITOCHONDRIA OF <i>NEUROSPORA CRASSA </i>

Kuriyama, Yoshiaki; Luck, David

doi:10.1083/jcb.59.3.776

Cited by 36 publications

(5 citation statements)

References 22 publications

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“…In these cases some of the organellar (33,118,200) or procaryotic ribosomes (214) have been reported to be bound, via their nascent polypeptides, to the inner organellar membranes . The products of such membranebound ribosomes could be transferred to the intermembrane or periplasmic spaces or be inserted into the inner or outer organellar or bacterial membranes .…”

Section: Vectorial Discharge Of Nascentmentioning

confidence: 99%

Mechanisms for the incorporation of proteins in membranes and organelles.

Sabatini¹,

Kreibich²,

Morimoto³

et al. 1982

The Journal of Cell Biology

869

281

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Section: Vectorial Discharge Of Nascentmentioning

confidence: 99%

Mechanisms for the incorporation of proteins in membranes and organelles.

Sabatini¹,

Kreibich²,

Morimoto³

et al. 1982

The Journal of Cell Biology

869

281

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“…The recommended incubation of thylakoid membranes with RNases in the absence of Mg2' and with 5 mm EDTA present (Fig. 1) is designed to induce unfolding of the ribosomes structure (13,19) and so cause maximal exposure of RNA to the added enzymes.…”

Section: Discussionmentioning

confidence: 99%

A Method for Enzymic Extraction and the Measurement of Chloroplast RNA

Fish

Jagendorf²

1980

Plant Physiol.

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Smillie and Krotkov (23) compared various methods for quantitating RNA in plant materials, and concluded that the SchmidtThannhauser procedure (20), which involves repeated tissue extractions with acid and organic solvents followed by KOH hydrolysis and use of an anion exchange resin, is the most reliable method for the widest range of plant tissues. De Deken-Grenson and de Deken (9) made the same conclusion, and described a batch method for the use of Dowex 2 in purifying the free nucleotides resulting from KOH hydrolysis of the RNA obtained by the Schmidt-Thannhauser procedure. However, the number of manipulations and the amount of sample required in the procedure described by Smillie and Krotkov (23) on the enhancement by high mol wt RNA of ethidium bromide fluorescence to estimate bound ribosomes in pea chloroplasts.Here, we describe a method for the extraction and measurement of thylakoid-associated RNA which does not require extraction or purification of undegraded RNA. Ribonucleases are added to release the thylakoid-associated RNA into the supernatant as nucleotides and oligonucleotides, thus reducing sample manipulation to a minimum. Miniature Dowex l-X2 columns are used to remove materials which interfere with analysis by UV A or the orcinol reaction for pentose.MATERIALS AND METHODS Dowex 1-X2 resin and Econo-column reservoirs were purchased from Bio-Rad. Hepes, EDTA (tetrasodium salt), pancreatic RNase T1, Torula yeast, RNA (type VI), pronase (protease type VI from Streptomyces griseus), p-aminosalicylate, and Tris base were purchased from Sigma; 8-hydroxyquinoline from Sargent-Welch; and triisopropylnaphthalene sulfonic acid sodium salt from Eastman Organic Chemicals. Pea (Pisum sativum var. Progress No. 9) seeds (captan-coated) were purchased from Agway.Columns were made by breaking off Pasteur pipettes to a final length of 10 cm, and then attaching Econo-column reservoirs at their upper ends with latex tubing and a 2.5-cm section of glass tubing. Filter paper discs (VWR No. 2831080 filter paper) cut with a No. 2 cork borer were forced to the bottom of the column with a 4-mm glass rod. A holder accommodating 30 columns were made from a 2.54-cm Plexiglas slab so that the holes for the columns were aligned with those of a test tube rack.Peas were grown for 12-16 days in Vermiculite in flats in a controlled environment chamber on a 16-h day/8-h night or a 12-h day/12-h night schedule at 21-23 C.Pea shoots were homogenized in a buffer containing 0.2 M sucrose, 200 mi Tris-HCl (pH 8.5), 60 ms KCI, and 15 mi Mgacetate. Isolation of thylakoids was performed as described by Yamamoto and Jagendorf (in preparation), and included an initial centrifugation, resuspension, and centrifuging through a washing layer of 0.5 M sucrose, collection of membranes at the lower interface following a sucrose step-gradient centrifugation (resuspended pellet without sucrose over 0.5 and 1.8 M sucrose steps), and a final washing by centrifugation. Between each step the resuspended pellets were disrupted by passing four times...

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“…The quantification of polysomal mRNAs is commonly used as a proxy for translational activity [ 21 , 22 ]. However, the firm association of mitochondrial ribosomes to the inner membrane [ 19 , 20 , 23 , 24 ] complicates mitochondrial polysomal mRNA quantification.…”

Section: Introductionmentioning

confidence: 99%

Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

et al. 2017

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BackgroundTo investigate translational regulation of gene expression in plant mitochondria, a mitochondrial polysome isolation protocol was established for tobacco to investigate polysomal mRNA loading as a proxy for translational activity. Furthermore, we developed an oligonucleotide based microarray platform to determine the level of Nicotiana tabacum and Arabidopsis thaliana mitochondrial mRNA.ResultsMicroarray analysis of free and polysomal mRNAs was used to characterize differences in the levels of free transcripts and ribosome-bound mRNAs in various organs of tobacco plants. We have observed higher mitochondrial transcript levels in young leaves, flowers and floral buds as compared to fully expanded leaves and roots. A similar pattern of abundance was observed for ribosome-bound mitochondrial mRNAs in these tissues. However, the accumulation of the mitochondrial protein COX2 was found to be inversely related to that of its ribosome-bound mRNA.ConclusionsOur results indicate that the association of mitochondrial mRNAs to ribosomes is largely determined by the total transcript level of a gene. However, at least for Cox2, we demonstrated that the level of ribosome-bound mRNA is not reflected by the amount of COX2 protein.Electronic supplementary materialThe online version of this article (10.1186/s13007-017-0257-4) contains supplementary material, which is available to authorized users.

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MEMBRANE-ASSOCIATED RIBOSOMES IN MITOCHONDRIA OF NEUROSPORA CRASSA

Cited by 36 publications

References 22 publications

Mechanisms for the incorporation of proteins in membranes and organelles.

Mechanisms for the incorporation of proteins in membranes and organelles.

A Method for Enzymic Extraction and the Measurement of Chloroplast RNA

Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

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