Stable and Labile Products of Mitochondrial Protein Synthesis <i>in vitro</i>

Wheeldon, L. W.; Dianoux, Anne-Christine; Bof, Mireille; Vignais, Pierre V.

doi:10.1111/j.1432-1033.1974.tb03611.x

Cited by 52 publications

(26 citation statements)

References 46 publications

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“…Although Tzagoloff and Akai [28] and Wheeldon et al [39] have reported single low-molecular-weight moieties in yeast and rat liver, respectively, the data of Kuntzel and Blossey [38] and the higher resolution allowed by the autoradiographic procedure used here shows two labelled components. The bands as they appear on the gels of Tzagoloff and Akai and Wheeldon et al are broad and possibly could be resolved into two bands.…”

Section: Analysis Of the Products Of Mitochondria1 Protein Synthesismentioning

confidence: 47%

Biogenesis of Mitochondrial Membranes in Neurospora crassa

Hawley

Greenawalt

1975

European Journal of Biochemistry

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The conidia of Neurospora crassa entered logarithmic growth after a 1‐h lag period at 30°C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3‐h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide‐sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0–8‐h‐old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 μg cycloheximide/ml, increased from low levels in the conidia to peak levels at 3–4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0–4‐h‐old cells, but declined after 15 min of incorporation in 6 and 8‐h‐old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12200 to 80000). Within the high‐molecular‐weight class, proteins of between 12000 and 21500 molecular weight were preferentially labelled early in germination, whereas after 8 h of growth proteins of 27500 to 80000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000‐molecular‐weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.

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Section: Analysis Of the Products Of Mitochondria1 Protein Synthesismentioning

confidence: 47%

Biogenesis of Mitochondrial Membranes in Neurospora crassa

Hawley

Greenawalt

1975

European Journal of Biochemistry

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“…The same holds for chloramphenicol, an inhibitor of mitochondrial translation (Wheeldon et al 1974;Kalnov et al 1979b;Black-Schaefer et al 1991), and hemin . It is unclear at present why chloramphenicol should inhibit degradation, as other inhibitors of translation do not have this effect .…”

Section: Characterization Of the Proteolytic Systemmentioning

confidence: 79%

“…Black-Schaefer et al (1991) also observed two pools with different rates of turnover, one with a half-life of minutes, the other with a half-life of hours. Instability of (a subset of) mitochondrial translation products has also been observed in rat liver mitochondria (Wheeldon et al 1974;Desautels and Goldberg 1982b), as well as in HeLa cells and rat hepatoma cells in the presence of cycloheximide (Constantino and Attardi 1977;Hall and Hare 1990). As assembly of mitochondrially encoded subunits of inner membrane complexes depends on the presence of imported subunits, and these imported subunits will become limiting at some point in isolated mitochondria or when cytoplasmic translation is inhibited, the rapidly degraded mitochondrial translation products probably represent unassembled subunits.…”

Section: Degradation Of Mitochondrial Translation Productsmentioning

confidence: 92%

The role of protein degradation in mitochondrial function and biogenesis

1996

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“…Furthermore, such "precursor" polypeptides seem to be soluble in chloroform-methanol [8,28]. Thus it is well possible that mitochondrial proteins soluble in chloroform -methanol contain short-lived precursors, but from the data shown here it is clear that at least the proteins of peak1 are stable, mature products of intramitochondrial protein synthesis.…”

Section: Discussionmentioning

confidence: 93%

Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in Neurospora crassa

Küntzel

Pienia̧żzek

Pienia̧źek

et al. 1975

European Journal of Biochemistry

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Mitochondrial proteins soluble in neutral chloroform -methanol (2 : 1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak 11). Residual phospholipids are found only in peak 11. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid.Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak I1 proteins by 95 %. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70 % and 95 % respectively, but do not affect labelling of peak I1 proteins.At least 30 % of the translation products of mitochondrial DNA in vitro behave like peak I proteins :they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak I1 are encoded by nuclear genes and synthesized on cytoplasmic ribosomes.Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.Proteins soluble in organic solvents are generally believed to penetrate into the lipid bilayer of biomembranes and are therefore called "integral" or "intrinsic" membrane proteins [l, 21. Most proteins synthesized within mitochondria belong to this class because they are soluble either in acid or in neutral chloroform -methanol (2 : 1) [3 -81. Those mitochondrial translation products that are soluble in neutral chloroform -methanol have rather low molecular weights of around 8000 [3,6,8] and include a component of the "membrane fraction" of oligomycinsensitive ATPase in yeast [9]. A protein fraction of similar size and solubility properties has been synthesized in vitro in cell-free systems programmed with mitochondrial DNA or mitochondrial messenger RNA from Neurospora crassa [lo].This suggests that at least some mitochondrial genes code for small hydrophobic proteins, which penetrate the lipid phase of the inner mitochondrial membrane. However, this group of proteins could be quite heterogeneous and it is still open as to how many species are contributed by the mitochondrial or by the nuclear : cytoplasmic genetic system. In order to answer these questions and to study structure and function of individual intrinsic membrane proteins we have started to isolate and purify from Neurospora crassa mitochondrial proteins soluble in neutral chloroform -methanol (2 : 1).It turned out that most of these proteins are in a rather narrow molec...

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Stable and Labile Products of Mitochondrial Protein Synthesis in vitro

Cited by 52 publications

References 46 publications

Biogenesis of Mitochondrial Membranes in Neurospora crassa

Biogenesis of Mitochondrial Membranes in Neurospora crassa

The role of protein degradation in mitochondrial function and biogenesis

Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in Neurospora crassa

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