Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions.

TitleCharacterization of SURF-1 expression and Surf-1p function in normal and disease conditions.
Publication TypeJournal Article
Year of Publication1999
AuthorsTiranti, V, Galimberti, C, Nijtmans, L, Bovolenta, S, Perini, MP, Zeviani, M
JournalHum Mol Genet
Volume8
Issue13
Pagination2533-40
Date Published1999 Dec
ISSN0964-6906
KeywordsAnimals, Blotting, Western, COS Cells, DNA, Mitochondrial, Electron Transport Complex IV, Electrophoresis, Gel, Two-Dimensional, Genetic Complementation Test, Humans, Leigh Disease, Membrane Proteins, Mitochondria, Mitochondrial Proteins, Mutagenesis, Site-Directed, Mutation, Proteins, Recombinant Proteins, Saccharomyces cerevisiae Proteins
Abstract

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 against a 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 approximately 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 electrophoresis 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.

DOI10.1093/hmg/8.13.2533
Alternate JournalHum. Mol. Genet.
Citation Key10.1093/hmg/8.13.2533
PubMed ID10556302
Grant List1180 / / Telethon / Italy