Synthesis of mitochondrial proteins in isolated rat hepatocytes

Gellerfors, Pär; Wielburski, Antek; Nelson, Bruce R.

doi:10.1016/0014-5793(79)81201-6

Cited by 19 publications

(7 citation statements)

References 18 publications

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“…Mitochondria were isolated using a modification of the method of Gellerfors and Nelson (17). Cells from each culture plate were washed twice with 4OC 0.02 M sodium phosphate (pH 7.4) containing 0.14 M NaCl (PBS) and scraped with a rubber policeman into 25 mM Tris-HC1 (pH 7.4) containing 2 mM EDTA and 250 mM sucrose (isolation buffer).…”

Section: Isolation Of Mitochondriamentioning

confidence: 99%

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

Lund¹,

Ziola²

1986

Biochem. Cell Biol.

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How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Isolation Of Mitochondriamentioning

confidence: 99%

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

Lund¹,

Ziola²

1986

Biochem. Cell Biol.

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“…An additional 2.5 ml of methionine-free media was added to each plate. Cells to be treated with chloramphenicol and cycloheximide were adjusted to final concentrations of 0.05 and 0.5 mM, respectively (10) while the remaining one-third of the culture plates served as the control. [35S] methionine ( l0 #Ci/plate) was then added and the culture plates were incubated at 35"C in a 5% CO2 atmosphere and 90% humidity.…”

Section: Determination Of the Site Of Synthesis Of P16mentioning

confidence: 99%

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

Pavco¹,

Tuyle²

1985

The Journal of Cell Biology

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The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCI isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr ~ 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNAbinding fragment (Mr ~ 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand ~X174 DNA, double strand (RF) ~Xl 74 DNA, or Escherichia coil ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand ¢X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.A previous study from our laboratory (1) identified P I6 (Mr ----16,000) as the single detectable polypeptide present in native mtDNA-protein complexes isolated from rat liver mitochondria. One function of the bound protein component was to stabilize the displacement loop (D-loop) structure by protecting the nascent strand of the D-loop from branch migration upon parental strand cleavage in vitro. In the accompanying paper (2), PI6 was found to bind exclusively to the displaced single strand of normal and expanded Dloops and to the single strand gap segment of molecules with the characteristics of/3-gapped circles. Thus, in addition to stabilization of the D-loop structure, P 16 probably functions at all stages of the asymmetrical replication cycle of mtDNA. Further evidence from that study indicated that there were an average of ~49 P16 molecules per mtDNA in the bound population composed predominantly of D-loop DNA.The work presented here describes the isolation of Pl 6 and a protease resistant, DNA...

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“…The labeling conditions for isolated hepatocytes and hepatoma cells were essentially identical. The cells were labeled in a medium [ 1 l] supplemented with unlabeled amino acid mixture and [35S]methionine (>lOOO Ci/mmol) as in [8,14]. In vitro incorporation of [35S]methionine by isolated mitochondria was performed as in [4].…”

Section: Elsevierlnorth-holland Biomedical Pressmentioning

confidence: 99%

The characterization of mitochondrial translation products in rat liver and rat hepatoma cells

et al. 1981

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Synthesis of mitochondrial proteins in isolated rat hepatocytes

Cited by 19 publications

References 18 publications

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

The characterization of mitochondrial translation products in rat liver and rat hepatoma cells

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