scholarly journals Mitochondrial ribosomal proteins developed unconventional mitochondrial targeting signals due to structural constraints

2021 ◽  
Author(s):  
Yury Bykov ◽  
Tamara Flohr ◽  
Felix Boos ◽  
Johannes M. Herrmann ◽  
Maya Schuldiner
Keyword(s):  
Ribosomal Proteins ◽  
Protein Targeting ◽  
Mitochondrial Protein ◽  
Nuclear Genome ◽  
Structural Data ◽  
Structural Constraints ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Targeting Signals

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Available proteomic and structural data as well as computational predictions indicate that up to 25% of MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We characterized a set of 15 yeast MRPs in vivo and showed that 30% of them use internal mitochondrial targeting signals. We isolated a novel internal targeting signal from the conserved MRP Mrp17 (bS6). The Mrp17 targeting signal shares some properties as well as import components with conventional MTS-containing proteins but is not reliably predicted indicating that mitochondrial protein targeting is more versatile than expected. We hypothesize that internal targeting signals arose in MRPs when the N-terminus extension was constrained by ribosome assembly interfaces.

scholarly journals Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

1989 ◽  
Vol 9 (4) ◽  
pp. 1611-1620 ◽  
Author(s):  
S R Ellis ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Protein Import ◽  
Amino Terminus ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Trna Methyltransferase ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Passenger Protein

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

scholarly journals Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

2005 ◽  
Vol 386 (2) ◽  
pp. 381-386 ◽  
Author(s):  
Tamara A. DAILEY ◽  
John H. WOODRUFF ◽  
Harry A. DAILEY
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Mitochondrial Protein ◽  
Leader Sequence ◽  
Erythroid Cell ◽  
Mitochondrial Targeting ◽  
Erythroid Precursor ◽  
Binding Motifs ◽  
Coproporphyrinogen Oxidase

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haem-binding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haem-regulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases

1989 ◽  
Vol 9 (4) ◽  
pp. 1611-1620
Author(s):  
S R Ellis ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Protein Import ◽  
Amino Terminus ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Trna Methyltransferase ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Passenger Protein

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

scholarly journals THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
Author(s):  
Paul M. Lizardi ◽  
David J. L. Luck
Keyword(s):  
Protein Synthesis ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Selective Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

scholarly journals Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

2008 ◽  
Vol 22 (4) ◽  
pp. 951-964 ◽  
Author(s):  
Goro Sasaki ◽  
Tomohiro Ishii ◽  
Pancharatnam Jeyasuria ◽  
Youngah Jo ◽  
Assaf Bahat ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Adrenal Cortex ◽  
Regulatory Protein ◽  
Artificial Chromosome ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Steroidogenic Cells ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

scholarly journals Mitochondrial protein targeting signals

1988 ◽  
Vol 16 (5) ◽  
pp. 709-710 ◽  
Author(s):  
GRAEME A. REID ◽  
MICHELLE E. WALKER
Keyword(s):  
Protein Targeting ◽  
Mitochondrial Protein ◽  
Targeting Signals

scholarly journals Novel Role of Calmodulin in Regulating Protein Transport to Mitochondria in a Unicellular Eukaryote

2013 ◽  
Vol 33 (22) ◽  
pp. 4579-4593 ◽  
Author(s):  
Abhishek Aich ◽  
Chandrima Shaha
Keyword(s):  
Leishmania Donovani ◽  
Protein Transport ◽  
Mitochondrial Protein ◽  
Unicellular Eukaryote ◽  
Mitochondrial Targeting ◽  
Content Type ◽  
Mitochondrial Translocation ◽  
Tryparedoxin Peroxidase

Lower eukaryotes like the kinetoplastid parasites are good models to study evolution of cellular pathways during steps to eukaryogenesis. In this study, a kinetoplastid parasite,Leishmania donovani, was used to understand the process of mitochondrial translocation of a nucleus-encoded mitochondrial protein, the mitochondrial tryparedoxin peroxidase (mTXNPx). We report the presence of an N-terminal cleavable mitochondrial targeting signal (MTS) validated through deletion and grafting experiments. We also establish a novel finding of calmodulin (CaM) binding to the MTS of mTXNPx through specific residues. Mutation of CaM binding residues, keeping intact the residues involved in mitochondrial targeting and biochemical inhibition of CaM activity bothin vitroandin vivo, prevented mitochondrial translocation. Through reconstituted import assays, we demonstrate obstruction of mitochondrial translocation either in the absence of CaM or Ca2+or in the presence of CaM inhibitors. We also demonstrate the prevention of temperature-driven mTXNPx aggregation in the presence of CaM. These findings establish the idea that CaM is required for the transport of the protein to mitochondria through maintenance of translocation competence posttranslation.

scholarly journals HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Shuping Zhang ◽  
Alejandra Macias-Garcia ◽  
Jacob C Ulirsch ◽  
Jason Velazquez ◽  
Vincent L Butty ◽  
...  
Keyword(s):  
Iron Deficiency ◽  
Mitochondrial Function ◽  
Ribosomal Proteins ◽  
Target Genes ◽  
Mitochondrial Protein ◽  
Erythroid Differentiation ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Underlying Mechanisms

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

scholarly journals The identification of mecciRNAs and their roles in mitochondrial entry of proteins

2019 ◽  
Author(s):  
Xu Liu ◽  
Xiaolin Wang ◽  
Jingxin Li ◽  
Shanshan Hu ◽  
Yuqi Deng ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Mitochondrial Protein ◽  
Nuclear Genome ◽  
Circular Rnas ◽  
Physiological Conditions ◽  
Mammalian Mitochondria ◽  
Protein Entry ◽  
Regulate Protein

AbstractMammalian mitochondria have small genomes encoding very limited numbers of proteins. Over one thousand proteins and noncoding RNAs encoded by nuclear genome have to be imported from the cytosol into the mitochondria. Here we report the identification of hundreds of circular RNAs (mecciRNAs) encoded by mitochondrial genome. We provide both in vitro and in vivo evidence to show that mecciRNAs facilitate mitochondrial entry of nuclear-encoded proteins by serving as molecular chaperones in the folding of imported proteins. Known components of mitochondrial protein and RNA importation such as TOM40 and PNPASE interact with mecciRNAs and regulate protein entry. Expression of mecciRNAs is regulated, and these transcripts are critical for mitochondria in adapting to physiological conditions and diseases such as stresses and cancers by modulating mitochondrial protein importation. mecciRNAs and their associated physiological roles add categories and functions to eukaryotic circular RNAs, and shed novel lights on communication between mitochondria and nucleus.

scholarly journals Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

2007 ◽  
Vol 73 (10) ◽  
pp. 3371-3379 ◽  
Author(s):  
Elke Zameitat ◽  
Gerald Freymark ◽  
Cornelia D. Dietz ◽  
Monika Löffler ◽  
Michael Bölker
Keyword(s):  
Biochemical Characterization ◽  
De Novo ◽  
Ustilago Maydis ◽  
Functional Expression ◽  
Cytostatic Drug ◽  
Dihydroorotate Dehydrogenase ◽  
Mitochondrial Targeting ◽  
Pyrimidine Nucleotides ◽  
Targeting Signal

ABSTRACT Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

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