MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8

A forwards genetic screen of a chemically mutated plant population identified mitochondrial RNA editing factor 10 (MEF10) in Arabidopsis thaliana. MEF10 is a trans-factor required specifically for the C to U editing of site nad2-842. The MEF10 protein is characterized by a stretch of pentatricopeptide repeats (PPR) and a C-terminal extension domain ending with the amino acids DYW. Editing is lost in mutant plants but is recovered by transgenic introduction of an intact MEF10 gene. The MEF10 protein interacts with multiple organellar RNA editing factor 8 (MORF8) but not with other mitochondrial MORF proteins in yeast two hybrid assays. These results support the model that specific combinations of MORF and MEF proteins are involved in RNA editing in plant mitochondria.     

The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana

RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.     

Two Related RNA-editing Proteins Target the Same Sites in Mitochondria of Arabidopsis thaliana

The facilitators for specific cytosine-to-uridine RNA-editing events in plant mitochondria and plastids are pentatricopeptide repeat (PPR)-containing proteins with specific additional C-terminal domains. Here we report the related PPR proteins mitochondrial editing factor 8 (MEF8) and MEF8S with only five such repeats each to be both involved in RNA editing at the same two sites in mitochondria of Arabidopsis thaliana. Mutants of MEF8 show diminished editing in leaves but not in pollen, whereas mutants of the related protein MEF8S show reduced RNA editing in pollen but not in leaves. Overexpressed MEF8 or MEF8S both increase editing at the two target sites in a mef8 mutant. Double mutants of MEF8 and MEF8S are not viable although both identified target sites are in mRNAs for nonessential proteins. This suggests that MEF8 and MEF8S may have other essential functions beyond these two editing sites in complex I mRNAs.     

The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain

In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.     

Approaches to defining dual-targeted proteins in Arabidopsis

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana . These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dual-targeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.     

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.     

Subcellular localization of two types of ferrochelatase in cucumber

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.     

PPR蛋白影响植物生长发育的研究进展

植物生长发育是一个极其复杂的生理生化过程,受内外因素共同作用。PPR蛋白是核基因编码的具有重复PPR基序的蛋白,分布广泛,在高等植物中数量巨大。PPR蛋白的靶标一般是线粒体和叶绿体中转录的RNA前体,多数可与MORF互作,参与线粒体和叶绿体基因的RNA编辑。PPR蛋白缺失的突变体植株多数呈现异常表型,影响植物的正常生长发育。本文就近年来发现的PPR蛋白结构、分布,与RNA编辑的关系,及其对植物生长发育的影响进行了综述。