scholarly journals 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

1988 ◽  
Vol 107 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
H Murakami ◽  
D Pain ◽  
G Blobel
Keyword(s):  
Heat Shock ◽  
Matrix Protein ◽  
Protein Import ◽  
Protein S ◽  
Yeast Mitochondria ◽  
Related Protein ◽  
Free System ◽  
In Vitro System ◽  
Cytosolic Factors

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

Phage DNA Directed Enzyme Synthesis in in vitro System from Yeast Mitochondria

1972 ◽  
Vol 238 (81) ◽  
pp. 74-76 ◽  
Author(s):  
DIETMAR RICHTER ◽  
PETER HERRLICH ◽  
MANFRED SCHWEIGER
Keyword(s):  
Enzyme Synthesis ◽  
Yeast Mitochondria ◽  
In Vitro System ◽  
Phage Dna

Enhancement by Activated Protein C of tPA-induced Lysis in a Cell-free System

1987 ◽  
Author(s):  
J C Fredenburgh ◽  
D Collen ◽  
M E Nesheim
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Plasminogen Activation ◽  
Free System ◽  
Cell Free System ◽  
Normal Plasma ◽  
Lysis Time ◽  
A Cell

The profibrinolytic activity of human activated protein C (APC) was studied in a cell-free system using human plasma. Normal and Ba+* citrate adsorbed human plasmas were dialyzed against 150mM NaCl, 20mM Hepes, pH 7.4 and diluted to an A280 of 16. Reactions were initiated by the addition of aliquots of plasma to cuvettes containing human melanoma tPA and human thrombin at final concentrations of 1 and 30nM, respectively. The effects of Ca+* and varying concentrations of APC on clotlysis times were examined by monitoring turbidity at 600nM while maintaining the temperature at 37°C. The lysis time, defined as the midpoint of turbidity change, was 128 min for normal plasma containing 10 mM Ca+* and showed progressive and saturable shortening to about 90 min at > 50nM APC. In the absence of Ca+*, lysis time was 55 min for normal plasma and did not shorten in response to APC. With Ba+* citrate adsorbed plasma, the lysis time was 82 min in the presence of 10mM Ca+*, and shortened to 42 min without Ca+*. APC had no effect on lysis time in Ba+* adsorbed plasma either with or without Ca+*. Both bovine and human APC were equally potent. Electrophoresis in DodSO4 and autoradiography of plasma samples containing 125I-labelled plasminogen indicated enhanced rates of plasminogen activation in the presence of APC. These data indicate that APC decreases lysis time in vitro at the level of plasminogen activation. This effect is dependent on Ca+* and may involve additional vitamin K-dependent protein ( s).

Tension-sensitive kinetochore phosphorylation in vitro

1998 ◽  
Vol 111 (21) ◽  
pp. 3189-3196 ◽  
Author(s):  
R.B. Nicklas ◽  
M.S. Campbell ◽  
S.C. Ward ◽  
G.J. Gorbsky
Keyword(s):  
Mammalian Cells ◽  
Chemical Change ◽  
Protein S ◽  
Living Cells ◽  
Mechanical Tension ◽  
In Vitro System ◽  
Substrate Protein ◽  
Meiotic Chromosomes ◽  
System A

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect's X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate's phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore's own kinase and phosphatase.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

scholarly journals Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378 ◽  
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

Gene expression during cold and heat shock in wheat

1991 ◽  
Vol 69 (5-6) ◽  
pp. 383-391 ◽  
Author(s):  
Jean Danyluk ◽  
Eric Rassart ◽  
Fathey Sarhan
Keyword(s):  
Heat Shock ◽  
Low Temperature ◽  
Winter Wheat ◽  
Freezing Tolerance ◽  
Spring Wheat ◽  
Triticum Aestivum L ◽  
Messenger Rnas ◽  
Free System ◽  
Heat And Cold Stress

Translatable messenger RNAs expression was compared in cold- and heat-stressed winter wheat (Triticum aestivum L. 'Fredrick' and 'Norstar') and spring wheat (T. aestivum L. 'Glenlea'). Polyadenylated RNA isolated from the crown and leaf tissues was translated in a wheat germ cell free system and the acidic and basic in vitro products were resolved by two-dimensional SDS–PAGE and autoradiography. The results showed that low temperature stress rapidly induced two groups of mRNAs. The first group was transient in nature and consists of 18 mRNAs that reached their highest levels of induction after 24 h of low temperature exposure and then decreased to undetectable levels. The second group consists of 53 mRNAs that were also induced or increased rapidly, but maintained their levels of expression during the 4 weeks required to induce freezing tolerance. Among those, at least 34 were expressed at higher levels in the freezing tolerant winter wheat compared with the less tolerant spring wheat. This suggests a possible relation between the expression of these mRNAs and the capacity of each genotype to develop freezing tolerance. In the case of heat shock, 50 mRNAs were induced or increased after 3 h at 40 °C. Among these, the expression of only six mRNAs was altered in a similar manner in the three genotypes by both treatments. The remaining mRNAs code for typical heat shock proteins which are different from those induced by low temperature. None of these mRNAs has been associated with the development of freezing tolerance. These results suggest that heat and cold stress are controlled by different genetic systems.Key words: wheat, mRNAs, proteins, low temperature, heat stress.

Tom20-mediated mitochondrial protein import in muscle cells during differentiation

2000 ◽  
Vol 279 (5) ◽  
pp. C1393-C1400 ◽  
Author(s):  
Janice Y. Grey ◽  
Michael K. Connor ◽  
Joseph W. Gordon ◽  
Masato Yano ◽  
Masataka Mori ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Matrix Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Muscle Cells ◽  
In Vitro Assays ◽  
Organelle Biogenesis ◽  
Outer Membrane Receptor ◽  
The Matrix

Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40–60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.

scholarly journals Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

1999 ◽  
Vol 10 (10) ◽  
pp. 3289-3299 ◽  
Author(s):  
Ursula Fünfschilling ◽  
Sabine Rospert
Keyword(s):  
Protein Import ◽  
Surface Proteins ◽  
Yeast Mitochondria ◽  
Nascent Polypeptide ◽  
C Terminus ◽  
Precursor Proteins ◽  
Nascent Chain ◽  
Stimulatory Factor ◽  
Import System

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

In Vitro Models That Support Adhesion Specificity in Biofilms of Oral Bacteria

1997 ◽  
Vol 11 (1) ◽  
pp. 33-42 ◽  
Author(s):  
R.P. Ellen ◽  
G. Lépine ◽  
P.-M. Nghiem
Keyword(s):  
Matrix Protein ◽  
Protein S ◽  
Specific Protein ◽  
In Vitro Models ◽  
Oral Bacteria ◽  
Host Cells ◽  
In Vitro Assays ◽  
Covalent Bonds ◽  
Enzyme Substrate Recognition

Adhesion to adsorbed pellicles and interspecies co-adhesion to form plaque biofilms involve selective interactions of bacterial adhesins with specific receptors. Our laboratory has devised in vitro assays for co-adhesion between Actinomyces naeslundii and Streptococcus oralis or Porphyromonas gingivalis on saliva-coated mineral and hexadecane droplet substrata. P. gingivalis structures significant for co-adhesion with A. naeslundii include surface vesicles and fimbriae. A family of arginine-specific cysteine proteinases in vesicles may be involved in adherence to bacteria, to host cells, and to matrix proteins. New research from several laboratories has found that such proteinases are processed from genes encoding polyproteins containing both proteinase and hemagglutinin domains. In addition to enzyme-substrate recognition, bacterial adhesion is often determined by specific protein-peptide and lectin-carbohydrate recognition. A. naeslundii - salivary proline-rich protein, S. gordonii - salivary a-amylase, and Treponema denticola - matrix protein recognition are examples of the former. Co-adhesion of A. naeslundii and S. oralis is an example of the latter. Lactose can selectively desorb A. naeslundii cells from mixed biofilms with S. oralis, a demonstration of the significance of specificity. Although non-specific forces are probably secondary to stereochemical fit in determining the selective range of surfaces that bacteria have evolved to recognize and bind, they probably help stabilize non-covalent bonds within aligned, complementary domains.

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