| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions
Introduction
Results
Import of Surf-1p in vivo
Surf-1 expression in mutant cell lines
Complementation assays with mutant variants of Surf-1p
SHY1 expression in human Surf-1p `null' mutant cells
mtDNA translation and COX assembly in Surf-1 mutants
Discussion
Materials And Methods
Antibodies
Import in vivo
Western blot analysis
Generation of mutated variants of SURF-1 cDNAs and GFP/HA-tagged SHY1 constructs
Cell transfections, COX cytochemistry and biochemistry
Preparation of crude mitochondrial fractions
mtDNA translation and two-dimensional PAGE
Acknowledgements
References
Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions
Received August 6, 1999; Revised and Accepted September 27, 1999
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 ~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.
INTRODUCTION
Leigh syndrome (LS; MIM 256000), or subacute necrotizing encephalomyelopathy, is an early-onset progressive neurodegenerative disorder characterized by predominant involvement of the CNS (1). Affected infants show severe psychomotor delay, cerebellar and pyramidal signs, dystonia, respiratory abnormalities, incoordination of ocular movements and recurrent vomiting. Focal symmetric lesions are found by magnetic resonance imaging (MRI) in the brainstem, thalamus and posterior columns of the spinal cord. Ragged red fibers, a common feature of mitochondrial disease, are consistently absent in skeletal muscle (2).
LS is a genetically heterogeneous entity. In some cases, it is attributable to mitochondrial DNA (mtDNA) mutations, in others to an autosomal recessive defect of a nuclear gene. In still other cases, the defect is X-linked or sporadic, as in the case of the defect of the E1[alpha] subunit of PDH. In all cases, all defects described to date in patients with LS affect the terminal oxidative metabolism and are likely to impair energy production (2,3). An isolated, severe and generalized defect of complex IV [cytochrome c oxidase (COX; EC 1.9.3.1)] is one of the most common biochemical abnormalities associated with LS (2-4). However, direct screening approaches failed to detect mutations in the COX subunit genes themselves (5). Very recently, a disease locus for LSCOX has been mapped to chromosome 9q34, and analysis of a candidate gene in the region, SURF-1, revealed deleterious mutations in most of the LSCOX patients investigated (6,7). Sequence analysis of SURF-1 in numerous LSCOX patients, as well as in patients with other forms of COX deficiency, has provided evidence that: (i) SURF-1 mutations are the most common cause of LSCOX; and (ii) the association between LS and SURF-1 mutations is highly specific, since no abnormalities of this gene were detected in COX defects not presenting with the clinical and neuropathological features of LS (8). However, both mutational analysis and complementation assays in cell culture also indicated that a proportion of LSCOX cases are not associated with mutations of SURF-1 (8), suggesting genetic heterogeneity of this condition. These conclusions are in agreement with early studies based on the identification of complementation groups in LSCOX (9,10). Although the precise function of SURF-1 remains to be elucidated, studies on the yeast homolog SHY-1 suggest that SURF-1 is involved in the maintenance of COX activity and mitochondrial respiration (11). To understand better the role of the product of SURF-1 (Surf-1p) and the pathogenesis of LSCOX, we have investigated the expression, mitochondrial targeting and possible interactions of Surf-1p with other components of the mitochondrial inner compartment in normal and disease conditions.
RESULTS
Import of Surf-1p in vivo
Figure 1A shows the results of immunoprecipitation of [35S]methionine-labeled proteins from COS-7 cells transfected with a recombinant human SURF-1 cDNA. The latter was fused in-frame with the nucleotide sequence encoding a strong epitope of the influenza virus hemagglutinin (HA). Using an anti-HA-specific monoclonal antibody, two protein species of ~35 and 31 kDa were immunoprecipitated from transfected cells. The first polypeptide is identical in size to the radiolabeled product of the HA-tagged SURF-1 cDNA translated in vitro, and corresponds to Surf-1pHA precursor. The second, shorter product corresponds to mature Surf-1pHA, resulting from the intramitochondrial cleavage of a leader peptide of ~42 amino acids. As expected, the leader peptide corresponds to the N-terminus of the precursor protein, since the mature protein still contains the HA epitope, which is fused with the protein C-terminus. As for most of the proteins imported into mitochondria, the import process is energy-dependent, since it is inhibited by treatment of transfected cells with valinomycin, an OXPHOS uncoupler.
Figure 1. Intracellular localization of Surf-1p. (A) In vivo import of Surf-1pHA expressed in COS-7 cells. Lane 1, immunoprecipitation from total [35S]methionine-radiolabeled proteins using an anti-HA monoclonal antibody; lane 2, immunoprecipitation from total radiolabeled proteins after incubation with 2 µM valinomycin; lane 3, immunoprecipitation of in vitro translated Surf-1pHA. (B) Sub-organellar localization of Surf-1p in rat liver mitochondria. M, mitochondrial fraction; Pt, pellet (mitochondrial membrane fraction); Sn, supernatant (soluble mitochondrial fraction). Percentages at the bottom indicate different concentrations of Na-deoxycholate (see text).
Figure 1B shows the results of sub-organellar localization of native Surf-1p in rat liver mitochondria. A band of ~30 kDa was immunostained by using AS182-196, a polyclonal antibody against amino acids 182-196, in the mid-portion of mature human Surf-1p. The identification of this band as corresponding to rat Surf-1p was demonstrated by an immunoadsorption test: no band was detected after pre-incubation of AS182-196 with peptide Y-16-Q (see Materials and Methods), the antigen used to produce the antibody (data not shown). Surf-1p cross-reacting material (CRM) was detected only in the membrane fraction of rat mitochondria, while virtually no protein was present in the soluble fraction, which largely corresponds to mitochondrial matrix. Treatment of the membrane fraction with increasing amounts of deoxycholate (DOC) resulted in the partial solubilization of rat Surf-1p. However, at a concentration as high as 5% DOC, ~20% of Surf-1p CRM was still detected in the membrane fraction.
Surf-1 expression in mutant cell lines
Figure 2A shows the results of northern blot analysis on total RNA using a 32P-radiolabeled full-length SURF-1 cDNA as a probe. In total RNA extracted from two control cell lines (Fig. 2A, lanes 1 and 2), two hybridization bands were visualized: a 1000 nucleotide hybridization band corresponding to the SURF-1 transcript, and a larger band, 2000 nucleotides in size, corresponding to 18S rRNA. The latter attribution was confirmed by the results of hybridization with an 18S rRNA-specific probe (data not shown). Only the 2000 nucleotide band was present in three SURF-1 mutant cell lines, whereas the 1000 nucleotide SURF-1-specific band was virtually absent (Fig. 2A, lanes 3-5). The three cell lines all carried loss-of-function mutations of SURF-1 in both alleles (8). One cell line (Fig. 2A, lane 3) was homozygous for the 37ins17 mutation, the second (lane 4) was homozygous for the 552delG mutation and the third (lane 5) was homozygous for the 751C->T mutation. However, in a total RNA sample from a fourth mutant cell line, a SURF-1-specific band was clearly present (Fig. 2A, lane 6). This cell line, called LSCOX772delCC derived from a patient in whom a frameshift mutation in exon 8 (mutation 772delCC) was found in a single allele, while the exons and the exon-intron boundaries belonging to the second allele resulted as normal (8). Western blot analysis was then performed on several fibroblast lysates, using AS182-196, an antibody specific to Surf-1p amino acids 182-196. To make possible the detection of specific protein products, western blot analysis was carried out on cell lines in which mutations of SURF-1 were located beyond the region encoding the 182-196 epitope. Examples of this analysis are shown in Figure 2B. No Surf-1p-specific band was detected in three cell lines homozygous for different `stop' mutations (lanes 3-5). The amino acid changes predicted by these mutations are Q251X, W288X and K290X, respectively. No Surf-1p CRM was also found in the `monoallelic' cell line LSCOX772delCC (lane 6). Identical results were obtained consistently in several other mutant cell lines carrying different SURF-1 mutations (data not shown).
Figure 2. (A) Northern blot analysis of total RNA from human cell lines using a SURF-1 cDNA as a probe. Lanes 1 and 2, control human cells (lane 1, 143B osteosarcoma cells; lane 2, normal human fibroblasts); lanes 3-5, fibroblast cell lines harboring different loss-of-function mutation of SURF-1 in both alleles (lane 3, homozygous mutation 37ins17; lane 4, homozygous mutation 552delG; lane 5, homozygous mutation 751C->T); lane 6, fibroblast cell line harboring the 772delCC mutation in a single allele. (B) Western blot analysis using the AS182-196 antibody. Lane 1, 143B osteosarcoma cells; lane 2, normal human fibroblasts; lanes 3-5, fibroblast cell lines harboring different loss-of-function mutation of SURF-1 in both alleles (lane 3, homozygous mutation 751C->T; lane 4, homozygous mutation 867G->A; lane 5, homozygous mutation 868insT); lane 6, fibroblast cell line harboring the 772delCC mutation in a single allele. An arrowhead indicates the band corresponding to wild-type Surf-1p. (C) RT-PCR analysis of SURF-1 full-length cDNAs (arrowheads) in a control cell line [#2, same as lane 2 in (A)] and in the monoallelic mutant cell line LSCOX772delCC [#6, same as lane 6 in (A)]. Markers (m) are expressed in kilobases (kb).
To investigate why no Surf-1p was detected in the `monoallelic' mutant cell line LSCOX772delCC, a SURF-1 cDNA was obtained by RT-PCR, and analyzed by agarose gel electrophoresis and nucleotide sequencing. Direct sequence analysis showed that the cDNA of LSCOX772delCC did not contain the 772delCC mutation. However, this cDNA appeared slightly larger than wild-type cDNA (Fig. 2C). Sequence analysis showed that the size abnormality was due to the insertion of 140 bp corresponding to the 5[prime] end of intron 2. The rearrangement predicts the synthesis of an aberrant and prematurely truncated protein, thus explaining the absence of Surf-1p CRM. This result was confirmed by several independent experiments, performed on LSCOX772delCC, and was specific to this cell line, since parallel RT-PCR amplifications performed in control cell lines produced a normal cDNA. Sequence analysis of the entire gene of this patient did not reveal the presence of any change either within or in proximity to exons, except for the heterozygous 772delCC mutation, nor was any obvious abnormality detected in intron sequences, including the sequence of intron 2.
Complementation assays with mutant variants of Surf-1p
We prepared three mutant SURF-1 cDNAs, containing stop codons in the same position or in close proximity to loss-of-function mutations found in our LSCOX patients (8). As shown in Table 1, the amino acid sequences that are predicted by these mutations are truncated variants of the Surf-1p precursor protein. The product of one variant (SURF-1STOP316) retains the first 105 amino acid residues on the N-terminal side of the wild-type sequence, that of the second (SURF-1STOP751) retains the first 250 amino acid residues and that of the third (SURF-1STOP868) retains the first 290 amino acid residues. Each of these variant cDNAs was inserted into a eukaryotic expression vector. COX activity was evaluated by biochemical (Table 1) and cytochemical (Fig. 3) assays after transient and stable transfections into a proband Surf-1p `null' cell line. The latter, called LSCOX37ins17, carries a homozygous frameshift mutation in exon 1 (37ins17) (8), which predicts the synthesis of a very short and aberrant protein product. Figure 3A shows an example of this analysis. No CRM corresponding to the protein product of SURF-1STOP751 was detected by AS182-196. The molecular weight of the predicted mature product of SURF-1STOP751 is ~21 kDa. As expected, no recovery of COX activity was obtained in this cell line. Biochemically (Table 1), the COX-specific activity in stable SURF-1STOP751 transfectants was 12.3 nmol/min/mg protein (normal values: 90.6 ± 12.2). Identical results were obtained by transfecting SURF-1STOP316 (COX-specific activity: 15.3 nmol/min/mg), but in this case we could not verify the presence of the corresponding protein because the SURF-1STOP316 mutation predicts the synthesis of a very short polypeptide which lacks the epitope specific to AS182-196. In contrast, a band of ~29 kDa, corresponding to the mature protein product of SURF-1STOP868, lacking only the 11 C-terminal amino acid residues, was clearly detected by AS182-196. However, in this case, too, no recovery of COX activity was present in transfected LSCOX37ins17 cells (COX-specific activity: 11.5 nmol/min/mg). As shown in Figure 3B, no phenotype rescue was obtained by transfecting a fourth construct, called [Delta]SURF-1162-306. The COX-specific activity measured in stable [Delta]SURF-1162-306 transfectants was 15.1 nmol/min/mg (Table 1). The protein product of [Delta]SURF-1162-306 lacks 48 amino acid residues in the region encompassing the conserved N-terminal domain (7,11) of the mature protein. The [Delta]SURF-1162-306 product still retains the first 42 N-terminal amino acid residues of the precursor Surf-1p sequence, which correspond to the putative leader peptide, plus the 12 adjacent amino acid residues of the mature protein. The N-terminal region of the Surf-1p precursor was left in the construct in order to allow the recombinant protein to be imported into, and processed within, the organelles. The presence of a band corresponding to the bona fide mature product of [Delta]SURF-1162-306 was revealed by using AS288-300, a polyclonal antibody against the Surf-1p C-terminus. The protein product detected in [Delta]SURF-1162-306-transfected cells was clearly shorter than the 35S-radiolabeled in vitro translation product of the same construct (Fig. 3B), indicating that the N-terminus of [Delta]Surf-1p had been processed in vivo.
Figure 3. Expression of mutant variants of Surf-1p in `null' mutant cells. (A) (Top) Western blot analysis using AS182-196 Surf-1+/+, control cell line; Surf-1-/-, LSCOX37ins17 mutant cells; Surf-1STOP751 and Surf-1STOP868, LSCOX37ins17 mutant cells after transfection with the SURF-1STOP751 and SURF-1STOP868 constructs (see text). Arrowheads indicate Surf-1p-specific signals. (Bottom) Cytochemical reaction to COX in the same cell lines. (B) (Top) Western blot analysis using AS182-196 Surf-1+/+, control cell line; [Delta]Surf-1162-306, LSCOX37ins17 mutant cells after transfection with [Delta]SURF-1162-306; i.v. [Delta]Surf-1162-306, [35S]methionine-labeled in vitro translation product of [Delta]SURF-1162-306. (Bottom) Cytochemical reaction to COX in the same cell lines.
Table 1. Recombinant cDNA constructs
| Construct | Mutation in recombinant cDNA | Naturally occurring mutation | Predicted recombinant protein | Expression of recombinant protein | COX activity in LSCOX37ins17 transfectants (nmol/min/mg)a |
| wt-SURF-1 | No | No | Wild-type Surf-1p | Yes | 72.6 |
| SURF-1STOP316 | TAA stop on codon 316-318 | 312del10insAT in exon 4 | P106X | ? | 15.3 |
| SURF-1STOP751 | TAG stop on codon 751-753 | 751C->T in exon 7 | Q251X | No | 12.3 |
| SURF-1STOP868 | TAG stop on codon 871-873 | 868insT in exon 9 | K291X | Yes | 11.5 |
| [Delta]SURF-1162-306 | Deletion of bp 162-306 | No | 54del48 | Yes | 15.1 |
| SHY1HAb | No | No | Shy1pHA | Yes | 15.0 |
bSHY1 is the yeast homolog of SURF-1.
In contrast to the results obtained by transfecting these mutant variants, complete restoration of COX activity (72.6 nmol/min/mg) was demonstrated after stable transfection of wt-SURF-1, a recombinant vector expressing the wild-type human SURF-1 cDNA (Table 1).
SHY1 expression in human Surf-1p `null' mutant cells
No rescue of COX phenotype was obtained by transfecting into our LSCOX37ins17 mutant cells the recombinant plasmid pSHY1HA (Fig. 4). The latter contains the full-length cDNA of SHY1, the yeast homolog of SURF-1. The COX-specific activity in stable pSHY1 transfectants was 15.0 nmol/min/mg (Table 1). As shown in Figure 4, the expression and mitochondrial localization of recombinant Shy-1 protein (Shy1p) was confirmed by transfecting into LSCOX37ins17 cells a chimeric construct called pSHY1HA/GFP (see Materials and Methods) expressing Shy-1p fused on the C-terminus with Aequora victoria green fluorescent protein (GFP).
Figure 4. Expression of Shy-1p in Surf-1p `null' mutant cells. (A) Direct GFP fluorescence (485 nm) after transfection with pSHY-1HA/GFP. (B) Rhodamine-specific immunofluorescence (546 nm) of the same cells using a polyclonal antibody against the mitochondrial single-stranded DNA-binding protein (mtSSB). (C) Cytochemical reaction to COX on LSCOX37ins17 mutant cells after transfection with pSHY-1HA.
mtDNA translation and COX assembly in Surf-1 mutants
Neither qualitative nor quantitative differences were detected between SURF-1 mutant and control cell lines (data not shown), suggesting that the absence of Surf-1p does not impair mtDNA translation in vivo.
To evaluate the effects of the absence of Surf-1p on the assembly of COX, experiments based on two-dimensional gel electrophoresis were performed in SURF-1 mutant and control cell lines. A specific anti-COXI monoclonal antibody was used to immunostain COX-specific subcomplexes (Fig. 5). Four subcomplexes (S1, S2, S3 and S4) previously have been defined as COX assembly intermediates (12). The identity of these subcomplexes was confirmed by using antibodies against COXII, COXIV and holo-COX (data not shown). In a control cell line, most of the COXI-specific CRM was confined to S4, corresponding to fully assembled COX. In contrast, most of the CRM in Surf-1p `null' mutants was present in subcomplexes S1 and S2. The altered COXI distribution of the mutant cell lines could be corrected by re-expressing a SURF-1 full-length cDNA (data not shown).
Figure 5. Western blot analysis of COXI on mitochondrial fractions electrophoresed through blue native two-dimensional gels. S1-S4, different COX assembly steps. (A) Control cell line. (B-D) SURF-1 `null' mutant LSCOX patients. (E) A SURF-1+/+ Leigh-like patient (8) with a severe reduction of COX activity. Arrows indicate the directions of the first and second electrophoretic runs.
DISCUSSION
To gain insight into the function of Surf-1p, we first verified whether Surf-1p is a mitochondrial protein. Using an in vivo import assay and western blot-based sub-organellar immunolocalization, we have shown that Surf-1p is synthesized as a larger precursor, imported into mitochondria via an energy-dependent pathway and cleaved from an N-terminal leader peptide. These results are in agreement with data based on immunofluorescence indicating the mitochondrial localization of Surf-1p (8). Like Shy-1p (11), mature Surf-1p is then stably integrated into the inner mitochondrial membrane, since the solubilization of the protein requires the use of detergent at concentrations normally used to solubilize intrinsic membrane proteins.
To evaluate the expression of SURF-1 in LSCOX, SURF-1 mRNA and Surf-1p were analyzed in patients carrying different mutations of the gene. All mutations predicted the loss of function of the protein (8). Northern blot and RT-PCR were used to analyze the expression of SURF-1 alleles carrying mutations in the 5[prime] end. No transcript was detected in different mutations spanning the first 770 nucleotides of SURF-1, suggesting that these mutations are associated with severe mRNA instability (13). Western blot analysis was used to test the presence and amount of Surf-1p CRM in cell lines carrying different mutations in the C-terminal half of the protein. No Surf-1p CRM was detected in any case, including cell lines carrying mutations in only one allele. In one of these `monoallelic' mutant patients, the mRNA carrying the loss-of-function mutation was virtually absent, further confirming the instability of these mutant transcripts. The only cDNA species found by RT-PCR corresponded to an aberrant SURF-1 mRNA containing a complex rearrangement most probably due to a splicing error. While this finding can explain the absence of Surf-1p CRM in this patient, and the resulting biochemical and clinical phenotypes, further work is necessary to understand the mechanism leading to the aberrant splicing of transcripts derived from apparently normal genes. LSCOX patients carrying `monoallelic' SURF-1 mutations are not rare (6-8), and in our experience all were characterized by the absence of Surf-1p CRM. The possibility of a `dominant-negative' effect of these mutations cannot be excluded in principle. However, this hypothesis is made very unlikely by both the structure of the pedigrees of these patients and the presence of the same mutations in the carrier parents of homozygous or compound heterozygous subjects in whom transmission of the trait is clearly recessive. Taken together, these results indicate that loss-of-function mutations of SURF-1 are associated with the absence of Surf-1p, due to either mRNA instability, rapid protein degradation or both. From a practical point of view, this observation suggests that characterization of Surf-1p should be included in the molecular diagnosis of LSCOX, especially in those patients for whom mutation analysis of the SURF-1 gene gives controversial or incomplete results.
To understand better whether and which domains of Surf-1p are essential for function, we generated several constructs containing truncated or partially deleted SURF-1 cDNAs. In three variants (SURF-1STOP316, SURF-1STOP751 and [Delta]SURF-1162-306), either one of the two highly conserved domains of Surf-1p (7,11) were missing. A fourth construct, SURF-1STOP868, was a recombinant variant which encodes a truncated protein lacking the last 11 amino acid residues of wild-type Surf-1p. The first three amino acid residues of this region are part of a conserved C-terminal domain of Surf-1p, while the remaining eight amino acid residues are not conserved phylogenetically. No rescue of COX phenotype was obtained in any of the cases. For SURF-1STOP751 and presumably also for SURF-1STOP316, this result reflects the complete absence of the corresponding product, due to either mRNA instability or rapid degradation of the mutant peptide. In contrast, we demonstrated that the protein product of [Delta]SURF-1162-306 could be expressed and processed into a mature protein in vivo. The absence of COX recovery in this case indicates that the N-terminal domain of mature Surf-1p is essential for function. This is in agreement with previous results obtained with mutant variants of SHY1 in yeast (11). Results from transfection of SURF-1STOP868 were also interesting. The protein encoded by SURF-1STOP868 is identical to that produced by a naturally occurring mutation (868insT). This mutation replaces the AAG codon encoding the K residue at position 291 of the Surf-1p precursor with a TAA stop codon (K291X). However, in contrast with the 868insT mutation, which is associated with the complete absence of the corresponding protein (Fig. 2B), SURF-1STOP868 is translated efficiently into a protein product. The latter appears to be processed into a mature form, whose size is slightly smaller than that of the corresponding wild-type form. Therefore, the maintenance of the COX defect in the SURF-1STOP868 transfectants cannot be attributed to the absence of the protein, but must be due to the loss of a crucial biological function associated with the integrity of the conserved C-terminal domain of Surf-1p. In contrast, the extension of the Surf-1p C-terminus does not impair its function, since we have shown previously (8) that expression of recombinant Surf-1pHA can rescue the COX phenotype of SURF-1 `null' mutant cells.
Based on the above considerations, it is perhaps not surprising that SHY1, the yeast homolog of SURF-1, failed to replace functionally the absence of Surf-1p. The SHY1 protein product appears to be expressed in human transfectants, and correctly targeted to mitochondria. Therefore, the absence of functional complementation is likely to reflect the relatively great divergence between the two amino acid sequences (only 23% amino acid identity) and suggests that Surf-1p and Shy1p both interact with co-evolved protein species.
Since Surf-1p is not part of the protein backbone of complex IV, one can hypothesize a role for Surf-1p in one of the several steps leading to the formation of a fully assembled, active holo-enzyme. Two distinct possibilities were tested: (i) a role in translation of mtDNA-encoded COX subunits; and (ii) a role in the assembly of the complex in the inner mitochondrial membrane.
Because there was no obvious difference in the mtDNA-specific translation products between several SURF-1 mutant and control cell lines, a role for Surf-1p in mtDNA translation is unlikely.
To test the hypothesis that Surf-1p plays a role in COX assembly, we investigated the accumulation of COX assembly intermediates by using blue native two-dimensional gel electrophoresis. COX is composed of 13 polypeptide subunits, three of which (COXI, COXII and COXIII) are encoded by mtDNA, translated within the inner compartment of mitochondria and directly inserted into the inner mitochondrial membrane. The 10 remaining nuclear-encoded subunits are synthesized in the cytosol and imported into mitochondria (14). Here they are inserted into the nascent complex in an ordered fashion. Four COX assembly intermediates can be detected (12). It has been proposed that these subcomplexes represent major steps in the COX assembly process. In the first step (S1), subunit I is inserted in the inner mitochondrial membrane, followed by the binding of heme a and heme a3 and a first copper atom (CuB). In the second step (S2), subunit IV is added to the nascent complex. A third step (S3) in the assembly process is believed to start with the binding of subunit II, which carries a copper pair (CuA), and subunit III, followed by subunits Va, Vb, VIc, VIIa, VIII, VIb and VIIc. In the final step (S4), a fully assembled holo-COX is obtained by the insertion of subunits VIIb and VIa. The results of our experiments, based on blue native two-dimensional gel electrophoresis, indicate that the absence of Surf-1p causes the accumulation of early intermediates S1 (COXI alone) and S2 (COXI + COXIV). Therefore, we conclude that Surf-1p is indeed a COX assembler, involved in the formation of subcomplex S3. It is likely that this involves the incorporation of subunit II into the COXI + COXIV intermediate, a crucial step which is believed to produce the rapid, `cascade-like' assembly of the other COX subunits. Moreover, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy of the COX assembly function of Surf-1p. It is interesting to note that the two-dimensional gel patterns obtained from the three Surf-1 mutant cell lines used in this experiment are virtually identical to each other. However, they clearly differ from the patterns obtained in both a normal control and a COX-deficient cell line not associated with SURF-1 mutations. The consistency of these results is in agreement with the observation that the clinical and biochemical features of Surf-1 mutant LSCOX patients are fairly homogeneous (8). In particular, the defect of COX activity appears to be the only OXPHOS abnormality in these patients, it is widespread in all tissues of the body, including skin fibroblasts, and it is quite severe, although a residual activity ranging from 5 to 20% can usually be found.
It is known from studies in yeast that assembly of COX can be impaired by mutations in factors involved in the assembly of the protein components of the complex, or by impairment of enzyme activities playing a role in the synthesis and intramitochondrial metabolism of prosthetic groups and co-factors. For instance, COX assembly is severely compromised in mutants of COX10, a farnesyl synthetase involved in the final steps of heme a synthesis (15), or in mutants of COX17, a protein involved in the metabolism of intramitochondrial copper (16). The current status of our knowledge on Surf-1p does not allow us to determine whether Surf-1p interacts directly or indirectly with COX subunits or with COX prosthetic groups. The observation that Shy-1p does not compensate the COX defect in Surf-1p `null' mutant cells is somewhat against the latter hypothesis, since the functional barrier among species is looser for genes encoding enzymes acting on non-protein substrates. Albeit largely circumstantial, these considerations favor a role for Surf-1p as a protein-interacting factor involved in the early steps of COX formation.
MATERIALS AND METHODS
Antibodies
The service offered by Neosystem (Strasbourg, France) was used to produce rabbit polyclonal antibodies against two oligopeptides specific to human Surf-1p. The first peptide encompasses the mid-portion of Surf-1p sequence, from amino acid 182 to 196 (peptide Y-16-Q); the second peptide encompasses the extreme C-terminus of the protein, from amino acid 288 to 300 (peptide Y-13-V). Both peptides were coupled to keyhole limpet hemocyanin, serving as an immunogenic carrier.
Rabbits were first immunized with an injection of the antigen-carrier conjugate, followed by two subsequent boosts, one every month. The two antisera, called AS182-196 and AS288-300, were collected from a final bleeding.
Import in vivo
The coding sequence of human SURF-1 cDNA was tagged on the 3[prime] end with the sequence encoding a strong antigenic epitope of the influenza virus HA. The molecular weight of the chimeric protein is ~35 kDa. The construct was inserted into the eukaryotic plasmid vector pcDNA3.1 (Invitrogen, Groningen, The Netherlands) and transfected in COS-7 cells as previously described (17). For in vivo mitochondrial targeting, total cellular proteins were radiolabeled with [35S]methionine for 2 h, in the presence or absence of 20 µM valinomycin. The specific translation products were immunoprecipitated using an anti-HA monoclonal antibody (Boehringer Mannheim, Mannheim, Germany) in the presence of Staphylococcus aureus lysate (Staph-A), and electrophoresed through a 12% SDS-polyacrylamide gel according to Laemmli (18). After fixation in 10% acetic acid, 25% isopropanol, the gel was washed for 15 min in Amplify (Amersham, Uppsala, Sweden), dried and autoradiographed overnight onto Hyperfilm (Amersham).
Western blot analysis
Approximately 2 × 106 cells were trypsinized, pelleted, sonicated, solubilized with 10 mM phosphate pH 7.2, 10 mM EDTA, 150 mM NaCl, 2% Triton X-100 and 0.25% SDS, in the presence of a mixture of protease inhibitors, and centrifuged at 100 000 g for 30 min at 4°C. A 100 µg aliquot of protein per lane was electrophoresed through an SDS-polyacrylamide gel. The gel was then electroblotted for 90 min onto a nitrocellulose filter. The latter was incubated with 5% non-fat milk in 20 mM Tris pH 8.0, 150 mM NaCl (TBS), 0.1% Tween-20 (MTT) for 60 min at room temperature followed by incubation overnight at 4°C with a 1:600 dilution of primary antibody or pre-immune serum in MTT. In some experiments, the primary antibody was pre-incubated overnight at 4°C with an excess of the antigen-carrier conjugate (immunoadsorption test). After four washings in TBS, 0.1% Tween-20 (5 min each) the filter was incubated for 60 min at room temperature with a 1:3000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) in MTT. After four additional washings, the peroxidase reaction was revealed by autoradiography using the chemiluminescence ECL kit (Amersham).
Generation of mutated variants of SURF-1 cDNAs and GFP/HA-tagged SHY1 constructs
Oligonucleotide-directed mutagenesis was used to generate truncated variants of SURF-1 cDNA. Briefly, the full-length SURF-1 cDNA was used as a template for PCR amplifications. A common forward primer covering the first ATG of the sequence was used in combination with different reverse primers containing a stop codon in the same position or in close proximity to loss-of-function mutations found in our LSCOX patients. The first construct (SURF-1STOP316) contained a TAA stop codon at nucleotide position 316-318, corresponding to mutation 312del10insAT in exon 4; the second construct (SURF-1STOP751) contained a TAG stop codon at nucleotide position 751-753, corresponding to mutation 751C->T in exon 7; the third construct (SURF-1STOP848) contained a TAG stop codon at nucleotide position 871-873, corresponding to mutation 868insT in exon 9.
A strategy based on overlap extension of PCR fragments (19), corresponding to different regions of SURF-1 cDNA, was used to create a mutant cDNA containing a deletion encompassing bp 162-306. This construct ([Delta]SURF-1162-306) encodes a protein which contains the putative leader peptide of Surf-1p, but lacks 48 amino acid residues in the N-terminal region of the mature protein.
Each construct was inserted into the eukaryotic vector pcDNA3.1 and expressed in a cell line harboring the 37ins17 mutation in exon 1 of SURF-1.
The SHY1 gene was isolated by means of PCR on chromosomal DNA from yeast strain FY1679-28C. By using PCR-generated restriction sites SpeI and NotI, the fragment was ligated into a yeast vector containing either the HA tag or the GFP-HA cassette. These contructs, called pSHY1HA and pSHYHA/GFP, were both able to complement an SHY1-disrupted yeast strain, indicating that no functional errors were introduced by PCR (data not shown). The fusion genes coding for both tagged proteins were isolated using suitable restriction sites (SpeI and XhoI) and ligated into pCDNA3.1.
Cell transfections, COX cytochemistry and biochemistry
All transfections were performed by electroporation of pCDNA3.1 constructs, as described (17). Stable transformants were obtained by continuous selection in Dulbecco's modified Eagle's medium containing 200 µg/ml of G418. COX activity was visualized cytochemically and measured spectrophotometrically as previously described (17).
Preparation of crude mitochondrial fractions
Cell pellets (~1 × 106/sample) harvested from exponentially growing cultures were resuspended in 100 µl of phosphate-buffered saline and incubated for 10 min on ice with digitonin (4 mg digitonin/ml fibroblast suspension). The samples were centrifuged for 1 min at 12 000 g and pellets were resuspended and washed once with PBS. The mitochondrial pellets were stored at -80°C until further processing. In one set of experiments, rat liver mitochondria were isolated as described elsewhere (20) and stored frozen at -80°C until performing the experiments.
mtDNA translation and two-dimensional PAGE
Translation of mtDNA-encoded polypeptides was performed as described (21).
For two-dimensional PAGE, crude mitochondrial pellets were resuspended in 100 µl of 1.5 M 6-aminohexanoic acid, 50 mM Tris pH 7.0. Twenty microliters of 10% [beta]-lauryl maltoside was added, and the samples were incubated for 15 min on ice. Clearing of the samples was performed by centrifugation at 12 000 g (20 min at 4°C). The supernatant was supplemented with 10 µl of 5% Serva Blue G in 1 M 6-aminohexanoic acid and used for the first dimension. Blue native two-dimensional PAGE was performed as described before (22,23). Gels were used for western blot analysis as described above. The monoclonal antibodies (Molecular Probes, Eugene, OR) used were 1D6-E1-A8 (raised against COXI), 12C4-F12 (raised against COXII) and 10G8-D12-C12 (raised against COXIV).
ACKNOWLEDGEMENTS
We are indebted to Ms Barbara Geehan for revising the manuscript, and to Dr Marina Mora, Mr Franco Carrara and Ms Andreina Bordoni for technical help. The financial support of Fondazione Telethon-Italy (grant no. 1168 to M.Z.), the Italian Ministry of Health (grant `Ricerca Finalizzata' ICS 030.3/RF98.37) and EMBO (short-term fellowship grant ASTF 9330 to L.N.) is gratefully acknowledged.
REFERENCES
+To whom correspondence should be addressed. Tel: +39 02 2394388; Fax: +39 02 2664236; Email: zeviani{at}tin.it
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