Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions.
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.
Tiranti
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Istituto Nazionale Neurologico 'C. Besta', Via Celoria 11, 20133 Milano, Italy.
Galimberti
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C
Nijtmans
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L
Bovolenta
S
S
Perini
M P
MP
Zeviani
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M
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005816:Genetic Complementation Test; D006801:Humans; D007888:Leigh Disease / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004272:DNA, Mitochondrial; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011994:Recombinant Proteins; C106291:SHY1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C092928:Surf-1 protein; D003576:Electron Transport Complex IV