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Title: Proteomic study of mitochondrial proteins
Author: Chen, R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Several proteins in bovine heart mitochondria with molecular masses of 29, 18 and 10 kDa have been demonstrated to be phosphorylated in a cAMP-dependent manner. The 18 kDa and 10 kDa proteins are subunits of complex I. In the presence of protein kinase A, subunits of purified complex I with the same molecular masses are phosphorylated in vitro. By Edman degradation and mass spectrometry of the radioactive protein bands from 32P-phosphorylated mitochondria and 32P-phosphorylated complex I, the 18kDa protein has been identified as subunit ESSS of complex I. It is phosphorylated at serine-20. In the 10 kDa band, subunit MWFE of complex I and subunit d of complex II are phosphorylated at serine-55 and serine-7, respectively. Five distinct 14-3-3 proteins have been detected in bovine heart mitochondria by immunological methods and by mass spectrometry. They are the b, g, e, h and ζ isoforms of the protein. Recombinant 14-3-3 b, g and ζ proteins have been over-expressed in Escherichia coli, and purified to homogeneity. The hydrophobic membrane protein, subunit c, has been isolated from bovine heart mitochondria, and from ovine and human lysosomal storage bodies associated with ceroid lipofuscinosis (Batten disease). By mass spectrometry, a post-translational modification with a mass of 42 Da is associated with a chymotryptic fragment of this protein in all three samples. By tandem MS sequencing, it has been shown that lysine-43 is trimethylated, and not acetylated, at the e-N-position of the residue is subunit c from all three samples. Therefore, trimethylation of subunit c is not, as has been suggested, a cause of abnormal accumulation of the subunit in lysosomes. This modification is not found in the equivalent lysine-44 of subunit c from yeast mitochondria. Therefore, the role of the modified lysine-43 in the assembly and (or) the functioning of the mitochondrial ATP synthase complex appears to be confined to higher organisms.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available