scholarly journals Yeast Mitochondrial Initiator tRNA Is Methylated at Guanosine 37 by the Trm5-encoded tRNA (Guanine-N1-)-methyltransferase

2007 ◽  
Vol 282 (38) ◽  
pp. 27744-27753 ◽  
Author(s):  
Changkeun Lee ◽  
Gisela Kramer ◽  
David E. Graham ◽  
Dean R. Appling
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Subcellular Fractionation ◽  
Mitochondrial Protein Synthesis ◽  
Reading Frame ◽  
Initiator Trna ◽  
Chromatography Analysis ◽  
Green Fluorescent

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m1G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m1G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Δtrm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNAMetf). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNAMetf and tRNAPhe. High pressure liquid chromatography analysis showed the methylated product to be m1G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1–33 (Δ1–33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Δ1–33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m1G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.

scholarly journals Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family

2003 ◽  
Vol 370 (1) ◽  
pp. 195-203 ◽  
Author(s):  
Liang LIANG ◽  
Mujun ZHAO ◽  
Zhenhua XU ◽  
Kazunari K. YOKOYAMA ◽  
Tsaiping LI
Keyword(s):  
Amino Acids ◽  
Cell Death ◽  
Small Intestine ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40kDa caspase-activated nuclease (DFF40) and a 45kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3α, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3α exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3α isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

scholarly journals MITOCHONDRIAL PROTEIN SYNTHESIS IN HELA CELLS

1974 ◽  
Vol 60 (3) ◽  
pp. 755-763 ◽  
Author(s):  
Jonas B. Galper
Keyword(s):  
Protein Synthesis ◽  
Ethidium Bromide ◽  
Mitochondrial Protein ◽  
Specific Protein ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Initiator Trna ◽  
Low Concentrations ◽  
Separate Protein

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a3 and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.

Isolation and incubation conditions to study heart mitochondrial protein synthesis

1990 ◽  
Vol 258 (3) ◽  
pp. E492-E502 ◽  
Author(s):  
E. E. McKee ◽  
B. L. Grier ◽  
G. S. Thompson ◽  
J. D. McCourt
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Adenine Nucleotide ◽  
Isolated Heart ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Specific Radioactivity ◽  
Companion Paper ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

scholarly journals An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

2004 ◽  
Vol 78 (3) ◽  
pp. 1488-1502 ◽  
Author(s):  
Thomas R. Jones ◽  
Shi-Wu Lee
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Cytoplasmic Vacuole ◽  
Viral Growth ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Green Fluorescent ◽  
And Function

ABSTRACT The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

scholarly journals The N-terminus of the serum- and glucocorticoid-inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover

2006 ◽  
Vol 399 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Arne Engelsberg ◽  
Franziska Kobelt ◽  
Dietmar Kuhl
Keyword(s):  
Amino Acids ◽  
Subcellular Localization ◽  
Half Life ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Mitochondrial Fraction ◽  
Mouse Tissue ◽  
N Terminus ◽  
Green Fluorescent

The serine/threonine protein kinase Sgk1 (serum- and glucocorticoid-inducible kinase 1) is characterized by a short half-life and has been implicated in the control of a large variety of functions in different subcellular compartments and tissues. Here, we analysed the influence of the N-terminus of Sgk1 on protein turnover and subcellular localization. Using green fluorescent protein-tagged Sgk1 deletion variants, we identified amino acids 17–32 to function as an anchor for the OMM (outer mitochondrial membrane). Subcellular fractionation of mouse tissue revealed a predominant localization of Sgk1 to the mitochondrial fraction. A cytosolic orientation of the kinase at the OMM was determined by in vitro import of Sgk1 and protease protection assays. Pulse–chase experiments showed that half-life and subcellular localization of Sgk1 are inseparable and determined by identical amino acids. Our results provide evidence that Sgk1 is primarily localized to the OMM and shed new light on the role of Sgk1 in the control of cellular function.

scholarly journals Selective synthesis of mitochondrial proteins by Chinese hamster ovary cells severely starved for various amino acids.

1984 ◽  
Vol 98 (4) ◽  
pp. 1603-1605 ◽  
Author(s):  
J W Chamberlain ◽  
J W Pollard ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Protein Synthesis Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

Chinese hamster ovary (CHO) cells were subjected to severe amino acid starvation for histidine, leucine, methionine, asparagine, tyrosine, glutamine, valine, and lysine, using amino acid analogs or mutations in specific aminoacyl-tRNA synthetases. At protein synthetic rates of less than 5%, in all cases, the newly synthesized proteins were found on two-dimensional electrophoretic gels to consist of a few intensely labeled spots, with the exception of lysine. This pattern could also be produced by strong inhibition of cytoplasmic protein synthesis with cycloheximide, and was abolished by preincubation with the mitochondrial protein synthesis inhibitor chloramphenicol. It appears therefore that the spots represent mitochondrial protein synthesis and that animal cells must have separate aminoacyl-tRNA synthetases for mitochondrial tRNAs corresponding to all these amino acids except, possibly, for lysine.

scholarly journals Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 674
Author(s):  
Francesco Capriglia ◽  
Francesca Rizzo ◽  
Giuseppe Petrosillo ◽  
Veronica Morea ◽  
Giulia d’Amati ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Mitochondrial Protein ◽  
Trna Synthetase ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Mitochondrial Functions ◽  
Carboxy Terminal

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

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