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Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells

Costantino, Paolo and Attardi, Giuseppe (1977) Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells. Journal of Biological Chemistry, 252 (5). pp. 1702-1711. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:COSjbc77

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Abstract

The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products.


Item Type:Article
Additional Information:Copyright © 1977 by the American Society for Biochemistry and Molecular Biology. (Received for publication, April 26, 1976) We wish to thank Edwin Ching for his help in some of the experiments described here. Dr. François Amalric for valuable discussions, Dr. James Hare for critically reviewing this manuscript, and Ms. Arger Drew and Ms. Gloria Engel for capable technical assistance. This work was supported by a Grant GM-11726 from the United States Public Health Service and by the Ente Nazionale Idrocarburi, Italy. Theoretical determination of the kinetics of accumulation of radioactivity in the mitochondrial protein products in the presence of emetine, as well as Figs. 5 to 9 and 11, is presented as a miniprint supplement immediately following this paper. For the convenience of those who prefer to obtain the supplementary material in the form of 13 pages of full size photocopies, it is available as JBC Document Number 76M-552.
Record Number:CaltechAUTHORS:COSjbc77
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:COSjbc77
Alternative URL:http://www.jbc.org/cgi/content/abstract/252/5/1702
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ID Code:10928
Collection:CaltechAUTHORS
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Deposited On:18 Jun 2008
Last Modified:26 Dec 2012 10:06

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