“Ping-Pong” Interactions between Mitochondrial tRNA Import Receptors within a Multiprotein Complex
01 Aug 2003-Molecular and Cellular Biology (American Society for Microbiology)-Vol. 23, Iss: 15, pp 5217-5224
TL;DR: By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNAIle to a 21-kDa component of the complex is dependent upon tRNATyr, whilebinding of tR NATyr to a 45-KDa component is inhibited by t RNAIle, suggesting this “ping-pong” mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.
Abstract: The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania, transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, approximately 640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of approximately 130-A particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNA(Tyr) as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNA(Tyr) and tRNA(Ile) were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNA(Ile) to a 21-kDa component of the complex is dependent upon tRNA(Tyr), while binding of tRNA(Tyr) to a 45-kDa component is inhibited by tRNA(Ile). This "ping-pong" mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.
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TL;DR: Results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.
Abstract: Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.
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Cites background or methods from "“Ping-Pong” Interactions between Mi..."
...Type I tRNAs bind to receptor RIC1, and anti-RIC1 antibodies inhibit this binding (11)....
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...Indicated amounts of recombinant protein and radiolabeled tRNA were incubated in 10mM Tris–HCl, pH 7.5, 10mM MgCl2, 2mM DTT and 0.1M KCl) for 30min on ice. Complexes were separated from unbound tRNA by 5–15% native PAGE (11) and detected by autoradiography....
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...Concentrated extract (8 ml=2 10(6) cell equivalent) was combined with recombinant RIC8A or its derivative (8 ng/2 ml) and phosphatidyl choline vesicles (50mg lipid in 10 ml) for 1 h at 48C before analysis by BN PAGE or import assay (11)....
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...We have previously purified a multisubunit complex (RNA Import Complex or RIC) from inner mitochondrial membranes of Leishmania tropica that actively transports tRNA across natural and artificial membranes (11)....
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...1M KCl, for 45min on ice and then electrophoresed on native 4–15% gradient polyacrylamide gels, as described (11)....
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01 Jan 2018
TL;DR: Development of human mitochondrial gene therapy strategies or plant mitochondrial biotechnologies suffer from the inability of conventional methodologies to genetically transform mitochondria, and a large set of alternative strategies aiming to target DNA or RNA into mitochondria are developed.
Abstract: Mitochondria resulted from an endosymbiosis event and subsequently kept their own genome. In the course of evolution, the mitochondrial DNA shrunk down but it still encodes essential components of the oxidative phosphorylation chain. Point mutations and deletions in the human mitochondrial DNA cause severe incurable neurodegenerative diseases and accumulate during aging. Rearrangements in the plant mitochondrial genome contribute to evolution and agronomical traits. Development of human mitochondrial gene therapy strategies or plant mitochondrial biotechnologies suffer from the inability of conventional methodologies to genetically transform mitochondria. The importance of these issues led to the development of a large set of alternative strategies aiming to target DNA or RNA into mitochondria, mainly in mammalian cells. A first group relied on natural RNA uptake pathways of mitochondria, using tRNA derivatives, tRNA mimics, 5S rRNA, and stem-loop structures of RNase P and RNase MRP RNAs as import shuttles, or taking a special RNA import complex as a carrier. Other strategies took advantage of the regular protein uptake pathway of mitochondria to design a series of DNA or RNA-binding plaforms driven to the organelles by mitochondrial targeting peptides. In a third category of approaches, elaborate DNA-binding lipophilic vesicles were rendered mitochondriotropic and served as carriers for organelle targeting. Finally, atypical protocols like hydrodynamic vein injection and magnetofection were adapted for the challenge. A number of these methodologies were claimed to be successful on the basis of functional or genetic observations, but there is still little consensus in the field.
References
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Book•
20 Dec 1983
TL;DR: The goal of this series is to pinpoint areas of chemistry where recent progress has outpaced what is covered in any available textbooks, and then seek out and persuade experts in these fields to produce relatively concise but instructive introductions to their fields.
Abstract: New textbooks at all levels of chemistry appear with great regularity. Some fields like basic biochemistry, organic reaction mechanisms, and chemical ther modynamics are well represented by many excellent texts, and new or revised editions are published sufficiently often to keep up with progress in research. However, some areas of chemistry, especially many of those taught at the grad uate level, suffer from a real lack of up-to-date textbooks. The most serious needs occur in fields that are rapidly changing. Textbooks in these subjects usually have to be written by scientists actually involved in the research which is advancing the field. It is not often easy to persuade such individuals to set time aside to help spread the knowledge they have accumulated. Our goal, in this series, is to pinpoint areas of chemistry where recent progress has outpaced what is covered in any available textbooks, and then seek out and persuade experts in these fields to produce relatively concise but instructive introductions to their fields. These should serve the needs of one semester or one quarter graduate courses in chemistry and biochemistry. In some cases the availability of texts in active research areas should help stimulate the creation of new courses. CHARLES R. CANTOR New York Preface This monograph is based on a review on polynucleotide structures written for a book series in 1976."
5,084 citations
TL;DR: The essential role of mitochondrial oxidative phosphorylation in cellular energy production, the generation of reactive oxygen species, and the initiation of apoptosis has suggested a number of novel mechanisms for mitochondrial pathology.
Abstract: Over the past 10 years, mitochondrial defects have been implicated in a wide variety of degenerative diseases, aging, and cancer. Studies on patients with these diseases have revealed much about the complexities of mitochondrial genetics, which involves an interplay between mutations in the mitochondrial and nuclear genomes. However, the pathophysiology of mitochondrial diseases has remained perplexing. The essential role of mitochondrial oxidative phosphorylation in cellular energy production, the generation of reactive oxygen species, and the initiation of apoptosis has suggested a number of novel mechanisms for mitochondrial pathology. The importance and interrelationship of these functions are now being studied in mouse models of mitochondrial disease.
2,950 citations
"“Ping-Pong” Interactions between Mi..." refers background in this paper
...Human mitochondria do not import tRNA, but a number of neuromuscular degenerative and metabolic diseases are caused by mutations in mitochondrial tRNA genes (21)....
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TL;DR: In this article, crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme, and the "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms.
Abstract: Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.
1,200 citations
TL;DR: Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins.
Abstract: Mitochondria import many hundreds of different proteins that are encoded by nuclear genes These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes Factors in the cytosol assist in targeting of preproteins Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins
1,079 citations
TL;DR: The TIM23 complex is a major translocase in the inner mitochondrial membrane that uses two energy sources, namely membrane potential and ATP, to facilitate preprotein translocation across the inner membrane and insertion into the inner membranes.
Abstract: Mitochondria comprise approx. 1000–3000 different proteins, almost all of which must be imported from the cytosol into the organelle. So far, six complex molecular machines, protein translocases, were identified that mediate this process. The TIM23 complex is a major translocase in the inner mitochondrial membrane. It uses two energy sources, namely membrane potential and ATP, to facilitate preprotein translocation across the inner membrane and insertion into the inner membrane. Recent research has led to the discovery of a number of new constituents of the TIM23 complex and to the unravelling of the mechanisms of preprotein translocation.
473 citations
"“Ping-Pong” Interactions between Mi..." refers background in this paper
...Moreover, the sequence and bioenergetic requirements for outer and inner membrane transfer are nonidentical (2), indicating the presence of a distinct transport machinery (the RNA import complex [RIC]) at the inner membrane, a situation similar to the TOM and TIM complexes for protein import (15)....
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