Journal Article

Characterization of <i>SURF-1</i> Expression and Surf-1p Function in Normal and Disease Conditions

Valeria Tiranti, Claudia Galimberti, Leo Nijtmans, Silvia Bovolenta, Maria Paola Perini and Massimo Zeviani

in Human Molecular Genetics

Volume 8, issue 13, pages 2533-2540
Published in print December 1999 | ISSN: 0964-6906
Published online December 1999 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/8.13.2533
Characterization of SURF-1 Expression and Surf-1p Function in Normal and Disease Conditions

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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.

Journal Article.  5507 words.  Illustrated.

Subjects: Genetics and Genomics

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