Characterization and biosynthesis of mitochondrial proteins

Gibb, Graham Morton (1985) Characterization and biosynthesis of mitochondrial proteins. PhD thesis, University of Glasgow.

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Mitochondria have their own very limited genetic system which codes for a few proteins of the inner membrane respiratory chain. As such, the bulk of mitochondrial proteins are nuclear encoded, synthesised in the cytoplasm on free ribosomes (in general as larger precursors) and subsequently imported into the correct compartment within the organelle. We were particularly interested in studying two mitochondrial proteins with respect to this problem of biosynthesis and import into the organelle. The first of these proteins, the phosphate transport protein is an integral, inner transmembraneous protein of mitochondria which has a major role to play in energy metabolism in the cell. It had only been partially characterized when we started to investigate it and as such this represented an interesting area of research. Purification of the phosphate transport protein from rat liver mitochondria was carried out by extraction in an 8% (v/v) Triton X-100 buffer and then adsorption chromatography on hydroxylapatite followed by Celite. SDS-polyacrylamide gel electrophoresis 10% (w/v) demonstrated that the protein band was apparently homogeneous when stained with Coomassie Blue and had an Mr of 34,000. However, when this gel was overlayed with 125 I-labelled concanavalin A to detect glycoproteins and exposed to autoradiography, numerous low and high Mr glycoprotein contaminants could be visualized. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer prior to purification. This initial extraction solubilised about 80% of the mitochondrial protein and effected quantitative removal of the contaminants. SDS-polyacrylamide gradient gel electrophoresis 14-20% (w/v) of the hydroxylapatite and Celite eluates revealed one major band of 34,000 when stained with Coomassie Blue. Densitometric scanning of the gel demonstrated that the protein was greater than 95% pure. This result was in marked contrast to the recent findings of several groups who have purified the phosphate transport protein from bovine and pig heart mitochondria, respectively. The hydroxylapatite eluates, when resolved on a similar gradient gel, demonstrated, four to five Coomassie Blue staining bands. In order to identify the protein, the sulphydryl group sensitivity of the phosphate transport protein was employed. Labelling studies with N-[3H]-ethylmaleimide showed that only the 34,000 Mr band was labelled in both the hydroxylapatite and, Celite fractions, when purified from rat liver mitochondria. This result was consistent with the known sulphydryl group sensitivity of the phosphate transport protein. Further confirmation has been provided by preliminary experiments with an antiserum directed against the 34,000 protein. Partial inhibition of mitoplast swelling in isotonic ammonium phosphate buffer was achieved when mitoplasts were incubated with the specific antiserum. To study the biosynthesis and import of this protein into mitochondria, in general, cultured mammalian cell lines were employed. The rabbit, polyclonal antiserum raised against the 34,000 Mr band proved to be unsuccessful when testing its specificity by immune-blotting and later with the in vivo mitochondrial import studies. Numerous polypeptides were observed when crude tissue-culture extracts were challenged with the antiphosphate transport protein serum and analysed by the immune-replica technique However, a preliminary experiment in the yeast rho- strain (rho) , which can accumulate large quantities of mitochondrial precursors in the presence of uncoupler, indicated that the protein was probably not. made as a larger Mr precursor. The antiserum raised against the rat liver mitochondrial phosphate transport protein strongly cross-reacted with a yeast protein of 29,000 under the above conditions. In addition the identity of this cross-reacting yeast protein has been partially provided by the immune-replica technique, where the antiserum specifically recognised a protein of 29,000 Mr in a yeast mitochondrial extract. The second protein of particular interest to us was the enzyme fumarase. This enzyme has been shown to be located in the cell cytosol as well as in the mitochondrial matrix. In the mitochondrial matrix, fumarase has a functional role to play in the operation of the 'citric acid cycle' and in the cell cytosol has an indirect role in the 'urea cycle', the tyrosine-oxidizing system and in purine biosynthesis. It has been reported in the literature that both forms of the enzyme are of the same Mr (subunit Mr of 49,000). In studying the biosynthesis of this enzyme, an additional problem to be overcome, is to determine the mechanism by which this bimodal distribution occurs.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology
Date of Award: 1985
Depositing User: Enlighten Team
Unique ID: glathesis:1985-76540
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:11
Last Modified: 19 Nov 2019 14:11

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