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

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.

Identification of a novel deletion in SURF1 gene: Heterogeneity in Leigh syndrome with COX deficiency

“…They have concluded that different mutations spanning the first 770 nucleotides of SURF1 lead to the absence of transcript. These results suggested that alterations in the initial region of the gene are probably associated with severe mRNA instability (Tiranti et al, 1999), which might be also occurring with the identified deletion SURF1 transcripts of the present work.…”

“…This duplication is predicted to generate a stop codon in amino acid 291 (p.Lys291X), resulting in a protein possibly smaller in 9 amino acids compared to the normal Surf1p. According to Tiranti et al (1999), this alteration generates a truncated protein with residual complex IV activity (Tiranti et al, 1999). The reported deficiency may be due to a loss of Surf1p conserved C-terminus transmembrane domain integrity.…”

“…The neuropathological diagnosis has been replaced by brain magnetic resonance imaging (MRI), which exhibits typical lesions (Tiranti et al, 1999). Usually, LS presents at an early age with psychomotor regression, pyramidal and extrapyramidal symptoms and signs of brain stem dysfunction, such as apnea and other respiratory rhythm abnormalities.…”

Post-transcriptional Silencing and Functional Characterization of theDrosophila melanogasterHomolog of HumanSurf1

“…Surf1 is ubiquitously expressed in human tissues, in particular in aerobic tissues such as heart, skeletal muscle, and kidney (Yao and Shoubridge 1999). The protein (SURF1), which is highly conserved from recent prokaryotes to humans (Poyau et al 1999), is characterized by an N-terminal mitochondrial targeting sequence and by two transmembrane domains and it is actively imported into mitochondria where it localizes to the inner membrane (Tiranti et al 1999a;Yao and Shoubridge 1999). More than 50 mutations of human 1 These authors contributed equally to this work.…”

“…COX is the terminal enzyme in both the eukaryotic and prokaryotic respiratory chain, catalyzing the reduction of molecular oxygen to water with concomitant proton pumping from the matrix to the intermembrane space. Studies in Surf1ÿ/ÿ cells from humans (Tiranti et al 1999a), mice (Agostino et al 2003), and yeast (Nijtmans et al 2001) indicate that SURF1 is involved in COX assembly. However, in human Surf1ÿ/ÿ cells low amounts of apparently fully assembled COX can still be detected by 2D-blue native electrophoresis, suggesting redundancy of the SURF1 function via unknown mechanism(s) (Coenen et al 1999;Tiranti et al 1999a;Hanson et al 2001).…”