Characterization of SURF-1 Expression and Surf-1p Function in Normal and Disease Conditions

• Published in: Human molecular genetics Vol. 8; no. 13; pp. 2533 - 2540
• Main Authors: Tiranti, Valeria, Galimberti, Claudia, Nijtmans, Leo, Bovolenta, Silvia, Paola Perini, Maria, Zeviani, Massimo
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.12.1999; Oxford Publishing Limited (England)
• Subjects: Animals; Biological and medical sciences; Blotting, Western; COS Cells; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; DNA, Mitochondrial - genetics; Electron Transport Complex IV - metabolism; Electrophoresis, Gel, Two-Dimensional; Genetic Complementation Test; Humans; Leigh Disease - enzymology; Leigh Disease - genetics; Leigh Disease - metabolism; Leigh syndrome; Medical sciences; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mitochondria - metabolism; Mitochondrial Proteins; Mutagenesis, Site-Directed; Mutation; Neurology; Proteins - genetics; Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae Proteins; SURF-1 gene; Surf-1 protein; Human; Nervous system diseases; Cell function; Enzyme; Leigh disease; Pathogenesis; Gene product; Cytochrome-c oxidase; Metabolic diseases; Molecular interaction; Gene expression; Enzymopathy; Cerebral disorder; Genetic disease; Mitochondria; Molecular assembly; Central nervous system disease; Genetics; Oxidoreductases; Mutation; Localization
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/8.13.2533

Loss-of-function mutations of the SURF-1 gene have been associated with Leigh syndrome with cytochrome c oxidase (COX) deficiency. Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown. Using antibodies againsta recombinant, hemagglutinin-tagged Surf-1p, we have demonstrated that this protein is imported into mitochondria as a larger precursor, which is then processed into the mature product by cleaving off an N-terminal leader polypeptide of ∼40 amino acids. By using western blot analysis with specific antibodies, we showed that Surf-1p is localized in and tightly bound to the mitochondrial inner membrane. The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position. Northern blot analysis showed the virtual absence of specific SURF-1 transcripts in different mutant cell lines. This result suggests that several mutations of SURF-1 are associated with severe mRNA instability. To understand better whether and which domains of the protein are essential for function, we generated several constructs with truncated or partially deleted SURF-1 cDNAs. None of these constructs, expressed into Surf-1p null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function. Finally, experiments based on blue native two-dimensional gel electro-phoresis indicated that assembly of COX in Surf-1p null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV. However, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy of this system.