ABSTRACT
A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.
Subject(s)
Amino Acid Transport Systems/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Mitochondria/metabolism , RNA Transport , RNA, Transfer/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation, Plant , Mitochondria/ultrastructure , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Protein Binding , Protein Biosynthesis , Protein Domains , RNA, Transfer/chemistry , RNA-Binding Proteins/metabolism , Species Specificity , Transcriptome/geneticsABSTRACT
Chloroplasts and mitochondria are unique endosymbiotic cellular organelles surrounded by two membranes. Essential metabolic networking between these compartments and their hosting cells requires the exchange of a large number of biochemical pathway intermediates in a directed and coordinated fashion across their inner and outer envelope membranes. Here, we describe the identification and functional characterization of a highly specific, regulated solute channel in the outer envelope of chloroplasts, named OEP40. Loss of OEP40 function in Arabidopsis thaliana results in early flowering under cold temperature. The reconstituted recombinant OEP40 protein forms a high conductance ß-barrel ion channel with subconductant states in planar lipid bilayers. The OEP40 channel is slightly cation-selective PK+/PCl- ≈ 4:1 and rectifying (iâ/iâ â 2) with a slope conductance of Gmax â 690 picosiemens. The OEP40 channel has a restriction zone diameter of â 1.4 nm and is permeable for glucose, glucose 1-phosphate and glucose 6-phosphate, but not for maltose. Moreover, channel properties are regulated by trehalose 6-phosphate, which cannot permeate. Altogether, our results indicate that OEP40 is a "glucose-gate" in the outer envelope membrane of chloroplasts, facilitating selective metabolite exchange between chloroplasts and the surrounding cell.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/chemistry , Chloroplast Proteins/chemistry , Chloroplasts/chemistry , Intracellular Membranes/chemistry , Membrane Proteins/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chloroplast Proteins/genetics , Chloroplast Proteins/metabolism , Chloroplasts/metabolism , Glucose/chemistry , Glucose/genetics , Glucose/metabolism , Intracellular Membranes/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids. Isolation of OEP16.2, a second OEP16 isoform from pea, in the current study allowed membrane localization and gene expression of OEP16 to be followed throughout seed development and germination of Arabidopsis thaliana and P. sativum. Thereby it can be shown on the transcript and protein level that the isoforms OEP16.1 and OEP16.2 in both plant species are alternating: whereas OEP16.1 is prominent in early embryo development and first leaves of the growing plantlet, OEP16.2 dominates in late seed development stages, which are associated with dormancy and desiccation, as well as early germination events. Further, OEP16.2 expression in seeds is under control of the phytohormone abscisic acid (ABA), leading to an ABA-hypersensitive phenotype of germinating oep16 knockout mutants. In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination. It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.
