Skip Navigation


Human Molecular Genetics Advance Access originally published online on November 27, 2007
Human Molecular Genetics 2008 17(6):775-788; doi:10.1093/hmg/ddm349
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
17/6/775    most recent
ddm349v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Fontanesi, F.
Right arrow Articles by Barrientos, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fontanesi, F.
Right arrow Articles by Barrientos, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Transcriptional activators HAP/NF-Y rescue a cytochrome c oxidase defect in yeast and human cells

Flavia Fontanesi1, Can Jin3, Alexander Tzagoloff3 and Antoni Barrientos1,2,*

1 Department of Neurology 2 Department of Biochemistry & Molecular Biology, The John T. MacDonald Foundation Center for Medical Genetics, University of Miami Miller School of Medicine, Miami, FL, USA 3 Department of Biological Sciences, Columbia University, New York, NY, USA

* To whom correspondence should be addressed at: Department of Neurology and Biochemistry & Molecular Biology, The John T. Macdonald Center for Medical Genetics, Universtiy of Miami Miller School of Medicine, 1600 NW 10th Ave., RMSB # 2067, Miami, FL 33136, USA. Tel: +1 3052438683; Fax: +1 3052433914; Email: abarrientos{at}med.miami.edu

Received October 29, 2007; Accepted November 26, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cell survival and energy production requires a functional mitochondrial respiratory chain. Biogenesis of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a very complicated process and requires the assistance of a large number of accessory factors. Defects in COX assembly alter cellular respiration and produce severe human encephalomyopathies. Mutations in SURF1, a COX assembly factor of exact unknown function, produce Leigh's syndrome (LS), the most frequent cause of COX deficiency in infants. In the yeast Saccharomyces cerevisiae, deletion of the SURF1 homologue SHY1 results in a similar COX deficiency. In order to identify genetic modifiers of the shy1 mutant phenotype, we have explored for genetic interactions involving SHY1. Here we report that overexpression of Hap4p, the catalytic subunit of the CCAAT binding transcriptional activator Hap2/3/4/5p complex, suppresses the respiratory defect of yeast shy1 mutants by increasing the expression of nuclear-encoded COX subunits that interact with the mitochondrially encoded Cox1p. Analogously, overexpression of the Hap complex human homologue NF-YA/B/C transcription complex in SURF1-deficient fibroblasts from an LS patient efficiently rescues their COX deficiency.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Eukaryotic cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain, catalyzes electron transfer from cytochrome c to molecular oxygen. COX is located in the mitochondrial inner membrane and is made up of some dozen different subunits encoded by both mitochondrial and nuclear DNA. The catalytic core of the enzyme consists of three subunits (Cox1p, 2p and 3p) all of which are mitochondrial gene products. Cox1p contains two redox centers, one formed by a low-spin heme a and the other by a binuclear center consisting of a high-spin heme a3 and CuB. A third redox center is formed by the two copper ions present in the CuA site of Cox2p. The 8 (yeast) or 10 (mammalian) nuclear encoded subunits act as a protective shield surrounding the catalytic core.

The subunits composing COX are thought to be matured and inserted into the nascent complex in an ordered fashion. At least four COX assembly intermediates (or subcomplexes) can be detected in human mitochondria, which represent major steps in the COX assembly process (1,2). Although more difficult to detect, COX subassemblies have also been described in some yeast mutants (3,4). Shy1p is an important COX assembly factor which functions early in the assembly pathway (5,6). Identifying the precise function of this protein is of considerable interest because mutations in its human homologue, SURF1, are responsible for most diagnosed cases of Leigh's syndrome (LS) presenting a COX deficiency, a devastating early-onset encephalomyopathy (7,8). Unlike other assembly-defective strains of yeast that display a complete absence of COX, shy1 mutants produce 10–15% fully assembled and functional COX (9), similar to LS patients who also have 10–30% of normal enzyme (7,8). This suggests that the function of Shy1p/SURF1 increases the efficiency of some step in the assembly process. Several functions have been proposed for Shy1p/SURF1 including (i) incorporation of subunit II into the nascent intermediates composed of subunit I plus subunit IV and V (yeast subunits 5a and 6) (2,10), (ii) assembly/stability of the heme a3 present in the binuclear center of Cox1p (6).

The COX deficiency of yeast shy1 mutants is rescued by mutations in Mss51p, a COX1 mRNA specific translational activator, which increase Cox1p expression in the shy1 mutants (5,11). Suppressor mechanisms may develop also in mammals and they could open a window for therapeutic interventions in LS patients. In line with this, SURF1 knockout mice have a mild mitochondrial disease and COX deficiency mainly affecting muscle and liver, and virtual absence of overt neurological symptoms (12).

To detect new genetic interactions of SHY1, we have searched for multicopy suppressors of the shy1 respiratory and COX defects. In this communication, we present evidence that HAP4, which codes for the catalytic subunit of the CCAAT binding transcriptional activator complex Hap2/3/4/5p, can act as a multicopy suppressor of shy1 mutants by over-expression of Cox5ap and Cox6p, which have been proposed to interact with Cox1p at early step of COX assembly. The mammalian homologue of the Hap complex is the ubiquitous transcriptional activator system NF-Y (13). Like Hap4p, over-expression of the catalytic subunit NF-YA rescues the COX activity defect of SURF1 mutant fibroblasts from an LS patient.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
HAP4 is a high copy suppressor of the shy1 mutants respiratory growth defect
In order to explore for genetic interactions involving SHY1, the C173/U1 strain carrying a point mutation in the SHY1 region coding for the first transmembrane domain of Shy1p (9) was used in a multicopy suppressor screen. Twenty respiratory competent clones were obtained with a yeast genomic multicopy plasmid library constructed in YEp24. With one exception all the clones contained plasmids with SHY1. The restriction map of pG91/T5, the single plasmid with an unrelated nuclear DNA insert, had end points between coordinates 229 300 and 240 578 on chromosome XI (Supplementary Material, Fig. S1). This plasmid was used to subclone the suppressor gene by transferring different regions of the insert to a yeast episomal vector and testing the ability of the new constructs to confer respiration in {Delta}shy1 cells. The suppressor activity mapped to a XmaI/PstI fragment containing only HAP4 (Supplementary Material, Fig. S1), coding for the catalytic subunit of the Hap complex (14). HAP4 acted as high copy suppressor of both, shy1 null and point mutant strains (Fig. 1A and Supplementary Material, Fig. S2). HAP4 transformants had a generation time in respiratory complete ethanol–glycerol containing media of 4.2 h compared with 28 h for the shy1 mutant and 2.4 h for the wild-type. Expression of HAP4 from an integrative plasmid did not rescue the respiratory growth defect (Supplementary Material, Fig. S2).


Figure 1
Figure 1
View larger version (79K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Characterization of shy1 mutants and HAP transformants. (A) The respiratory competent wild-type strain W303 and a {Delta}shy1 strain transformed with either empty plasmids (ep) or plasmids over-expressing genes for the different subunits of the HAP complex (HAP2, 3, 4 and 5) were grown over-night in liquid selective WO-D media. Ten-fold serial dilutions of the strains were plated on solid YPD or YPEG media and incubated at 30°C. Pictures were taken after 2 and 4 days of incubation, respectively. (B) KCN-sensitive endogenous cell respiration was measured polarographically in the presence of galactose in shy1 null ({Delta}shy1) and point mutants (W125 and C173) transformed with plasmids over-expressing the different subunits of the HAP complex. Where indicated, the genes were expressed from either integrative (i-) or episomal (e-) plasmids. (C) Total mitochondrial cytochrome spectra. The absorption bands corresponding to cytochromes a and a3 have maxima at 603 nm (a,a3). The maxima for cytochrome b (b) and for cytochrome c and c1 (c,c1) are 560 and 550 nm, respectively. (D) Oxygen consumption was assayed polarographically in mitochondria prepared from the indicated strains in the presence of either NADH or ascorbate-TMPD as substrates as described under Materials and Methods. (E) Mitochondrial respiratory chain enzyme spectrophotometric measurements in isolated mitochondria. Cytochrome oxidase (COX) and NADH cytochrome c reductase (NCCR) were measured as described under Materials and Methods. (F) Steady-state levels of COX subunits. Total mitochondrial proteins (30 µg) separated by 12% SDS–PAGE were transferred to nitrocellulose and probed with subunit-specific antibodies to COX subunits 1, 2, 4 and 5. The western blots were also probed with an antibody that recognizes a 45 kDa mitochondrial outer membrane protein (MOM45) to normalize the signals for protein loading. The bars indicate the mean ± SD from at least three independent sets of measurements.

 
The shy1 null and point mutant strains transformed with an empty plasmid respire at 15 or 20% of the wild-type rate, respectively, in whole cell assays performed in minimum media containing galactose. The KCN-sensitive respiratory activity is increased to 42–55% of wild-type in the shy1 null and point mutants over-expressing HAP4 (Fig. 1B). The presence of an additional chromosomally integrated copy of HAP4 in the {Delta}shy1 strain increased its respiratory capacity consistently from 15 to 20% (Fig. 1B) which is insufficient to produce a visible growth phenotype on solid YPEG (Supplementary Material, Fig. S2).

Partial restoration of shy1 respiratory growth defect by HAP4 over-expression results from an increase in COX
Spectra of mitochondrial cytochromes indicated the HAP4 over-expressors contain more ‘a’-type cytochromes than shy1 mutants (Fig. 1C). This was paralleled by a similar increase in overall NADH oxidase activity, which was 2.5-fold higher in the transformant and corresponded to 35–52% of the wild-type rate (Fig. 1D). The COX activity, which was measured either polarographically following the oxidation of ascorbate in the presence of TMPD (N,N,N’,N’-tetramethyl p-phenylenediamine) or spectrophotometrically by following the oxidation of ferrocytochrome c at 550 nm was also 2.5 times higher in the HAP4 over-expressors than in the shy1 mutants and ranged between 35 and 50% of wild-type values (Fig. 1D and E). Consistently, the steady-state concentrations of COX subunits in the HAP4 transformants were higher compared with the shy1 mutants (Fig. 1F).

Interestingly, the NADH cytochrome c reductase activity, which was previously reported to be higher in shy1 mutants (9), remained increased in the suppressed strains (Fig. 1E), and probably represents a compensatory mechanism of the partial COX defect.

Only the Hap4p component of the Hap2/3/4/5p complex restores the respiratory defect of shy1 mutants
Hap4p is the catalytic subunit of a multimeric transcription activator complex formed by three additional subunits, Hap2p, Hap3p and Hap5p. Interestingly only two of these subunits, Hap4p and Hap2p, have nuclear import sequences. Hap3p and 5p form a ternary complex with Hap2p in the cytoplasm and are imported together by a piggy-back mechanism (15). The ternary complex binds to the consensus sequences present in the promoter of their target genes and, upon binding of Hap4p to the complex, transcription is activated. HAP2, HAP3 and HAP5 genes are constitutively expressed, whereas HAP4 expression is highly regulated by carbon source: cells grown in media containing lactate, a respiratory substrate, have 4–5-fold more HAP4 mRNA than cells grown in media containing glucose (14,16).

We tested if over-expression of the Hap4p partner subunits could suppress shy1 mutants. No clearly detectable effect of HAP2, 3 and 5 on the ability of shy1 mutants to grow after 4 days on YPEG was noted (Fig. 1A). However, the respiratory activity of the null mutant and the W125 point mutant expressing HAP2 from a high copy plasmid increased from 15 to 20% and from 18 to 25% of the wild-type values, respectively (Fig. 1B). A possible explanation is that over-expression of HAP2 increases the amount of the ternary Hap2/3/5p DNA binding complex in the nucleus.

Suppression by HAP4 over-expression is not common among null mutants of COX assembly factors
Suppression of the respiratory growth defect of different COX assembly defective strains was assessed by transforming cox10, cox11, cox14, cox15, cox16, cox17, oxa1, mss51 and pet191 null mutants with HAP4 on a multicopy plasmid. A null mutant of cox5a was also included in the screening because, similar to {Delta}shy1 cells, it retains 15% of COX activity, in this case resulting from enzyme containing the isoform Cox5bp. In all cases tested, HAP4 over-expression failed to induce any suppression of their respiratory growth defect (data not shown), indicating that the suppression by HAP4 was specific to the shy1 null mutant, and suggesting a specific mechanism of HAP4 suppression in this strain.

The specificity of HAP4 suggested that it might act by either increasing the expression of a protein with an overlapping function with Shy1p or increasing the amount of specific COX subunits and/or assembly factors favoring a more efficient assembly of COX in the absence of Shy1p.

Suppression by HAP4 over-expression does not involve MSS51
Mutations in the COX1 mRNA specific translational activator MSS51 or over-expression of wild-type MSS51 suppresses shy1 null mutants by enhancing synthesis of Cox1p (5). HAP4 could indirectly affect Cox1p translation by increased expression of Mss51p. Northern blot analyses showed that the steady-state levels of MSS51 mRNA in {Delta}shy1 cells and in mutant cells over-expressing HAP4 were virtually identical (data not shown). Additionally, western blot analyses showed the amount of Mss51p in the mitochondrial membranes was not increased in shy1 mutants over-expressing HAP4 (Supplementary Material, Fig. S3).

Suppression by HAP4 could also be the result of a general increase in mitochondrial mass resulting in increased expression of mitochondrial proteins. HAP4 has been shown to stimulate mitochondrial biogenesis (17). This is supported by a significant increase in mitochondrial DNA nucleoids in wild-type and shy1 mutant cells over-expressing this transcription factor although the nucleoids are of smaller size (Supplementary Material, Fig. S4A and B). There is also a significant alteration in the morphology of the mitochondrial network which appears to be more fragmented, characteristic of conditions favoring mitochondrial biogenesis (Supplementary Material, Fig. S4C). Levels of mitochondrial DNA were estimated by Southern-blot analyses and were not significantly increased in wild-type or mutant cells overexpressing HAP4 (data not shown). Also, levels of COX1, and COX2 mRNAs, were not increased in cells overexpressing HAP4 as estimated by northern blot analyses of mitochondrial RNAs (Fig. 2A). Accordingly, in vivo translation assays did not show any significant increase of 35S-methionine incorporation into newly synthesized mitochondrial gene products in HAP4 over-expressing strains when cells were grown in galactose (Fig. 3A) and only a mild increase in cells submitted to a diauxic shift (Fig. 3B). The apparent discrepancy between the observed increase in mitochondrial nucleoids and the absence of obvious mitochondrial protein synthesis enhancement in cells over-expressing HAP4 can be explained by the absence of significant increase in mitochondrial DNA and RNA, and by HAP-independent factors such as Mss51p that may be limiting translation of Cox1p. These results also argue against an Mss51p-mediated mechanism of HAP4 suppression. Despite some variability intrinsic to in vivo mitochondrial protein synthesis experiments, the amount of newly synthesized Cox1p was higher in the HAP4 suppressor than in the mutant after 2 h of chase (Fig. 3B) suggesting that a larger amount of newly synthesized Cox1p is spared from proteolysis and can proceed in the assembly pathway.


Figure 2
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Effect of HAP4 overexpression on the steady-state levels of COX-related mRNAs. (A) Northern blots of total RNA probed for COX11, COX10, HEM2, COX4, COX5a, COX6 and ACT1. After processing, the membranes were exposed to X-ray film. In the lower panel, the images were digitalized and densitometry performed using the histogram function of the Adobe Photoshop program. The values were normalized by the signal of ACT1 as the loading control and expressed as the value relative to the control. (B) Northern blots of mitochondrial RNA extracts were hybridized with probes containing the entire coding sequence of cytochrome b (COB), subunits 1 (COX1) and 2 (COX2) and of 21S-rRNA as a loading control. The precursor (p) and mature (m) forms of COB and COX1 mRNAs are identified in the margin. In the lower panel the intensities of the bands quantified as in (A) were normalized by the 21S-rRNA signal and expressed as the value relative to the control. The values measured in two independent assays did not differ by more than 5%.

 


Figure 3
View larger version (58K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Additive effects of mss51 mutations and copper supplementation to the media on the shy1 suppression by HAP4 over-expression. (A) In vivo mitochondrial protein synthesis. Wild-type (WT), the null mutant {Delta}shy1 cells ({Delta}shy1) transformed with either an empty plasmid (–) or a plasmid over-expressing either HAP4 or HAP2, a spontaneous revertant of shy1 carrying a F199I mutation in MSS51 (5), and the revertant strain over-expressing HAP4, growing in minimum media containing 2% galactose and supplemented with the appropriate auxotrophic requirements were labeled with [35S]-methionine at 30°C for 15 min in the presence of cycloheximide. Equivalent amounts of total cellular proteins were separated by PAGE on a 17.5% polyacrylamide gel, transferred to a nitrocellulose membrane and exposed to an X-ray film. The mitochondrial translation products are identified on the left. (B) The same strains as in (A) plus a wild-type strain overexpressing HAP4 and a {Delta}shy1 strain overexpressing COX5a and COX6, were grown on minimum glucose media overnight and transferred to minimum ethanol/glycerol media during 4 h. Protein synthesis was performed as in (A). After 15 min pulse, 4 mg/ml puromycin and excess of cold methionine were added to stop the incorporation reaction and chase the synthesized products for the indicated times. To quantify the Cox1p signal images were digitalized and densitometry performed using the histogram function of the Adobe Photoshop program. The values measured in two independent assays did not differ by more than 10%. The values reported are averages of the two assays. (C) Endogenous cell respiration in cells grown in liquid WO-Gal media measured polarographically as described under Materials and Methods. (D) The same strains as in (A) were grown over-night in liquid selective WO-D media. Ten-fold serial dilutions of the different strains were plated on solid YPD, WO-EG, YPEG and YPEG media supplemented with 0.15% CuSO4 and incubated at 30°C. Pictures were taken after 2, 5 and 4 days of incubation respectively. (E) Same as in (C) with the exception that the different strains were grown in solid YPEG plates supplemented or not with 0.15% of CuSO4. Immediately before the assay, the cells were scraped from the plates and suspended in YPEG liquid media. The bars indicate the mean ± SD from at least three independent sets of measurements.

 
Suppression by HAP4 and MSS51 were found to be additive. A shy1 strain that reverted to a respiratory-competent phenotype by a spontaneous mss51F199I mutation (5) respired at 55% the wild-type rate. The comparable value for the HAP4 suppressed mutant was 40%. The respiratory activity of the mss51F199I revertant transformed with HAP4 was further increased to 85% (Fig. 3C). This strain grew on non-fermentable carbon sources (either YPEG or the more stringent WO-EG) significantly better than the HAP4 over-expressor or the shy1 revertant (Fig. 3D). These results suggest that the HAP4 suppression is not related to an increase in Cox1p synthesis but to enhancement of downstream events in the maturation/assembly of this and/or other COX subunits.

Copper and heme A are both prosthetic groups of Cox1p. Because Shy1p could be involved in maturation of Cox1p by contributing to the formation and/or stabilization of the CuB-heme a3 center, we tested if supplementation of the growth media with either hemin or copper induced any rescue of the respiratory defect observed in shy1 mutants. No effect on the growth of the shy1 null mutant strain was observed in the presence of hemin (not shown). As shown in Fig. 3D and Supplementary Material, Fig. S5A, respiratory media supplementation with 0.15% CuSO4 allowed a null mutant of shy1 to grow more efficiently although not at wild-type levels. The improved respiratory growth resulted from an increased cell respiratory capacity in YPEG from 20% of wild-type in the absence of Cu supplementation to almost 40% in the presence of the metal, a consequence of an increase in COX activity (Supplementary Material, Fig. S5B). The suppression by copper supplementation was no additive to either the suppression by mutations in mss51 or HAP4 over-expression (Fig. 3E).

Suppression by HAP4 over-expression does not involve over-expression of other COX assembly factors
Data derived from systematic analyses of yeast gene expression (17–19) summarized in Supplementary Material, Table S1, have revealed that although nuclear-encoded COX assembly factors are not co-regulated, some are directly or indirectly regulated by the Hap system.

In our system, HAP4 expression was under the control of its own promoter, which is repressed by glucose. Overexpression of HAP4 in a {Delta}shy1 mutant was induced in cells undergoing a diauxic shift by transferring them from galactose to ethanol–glycerol containing media for 4 h. Northern blot analyses allowed us to estimate HAP4 mRNA to be increased 5-fold in cells overexpressing HAP4 (data not shown). In these metabolic conditions, we assessed the steady-state mRNA levels of a series of candidate genes involved in COX biogenesis. We did detect a higher than 2-fold increase in the expression of COX10, a gene involved in heme a biosynthesis (Fig. 2B) and a consistent 1.5-fold increase in the amount of HEM2 mRNA, the gene encoding for the {delta}-aminolevulinate dehydratase, which catalyzes the conversion of delta-aminolevulinic acid to porphobilinogen, the second and rate-limiting step in the heme biosynthetic pathway and known to be regulated by the Hap system (20). Overexpression of these 2 genes in a shy1 mutant strain did not produce any suppression of its respiratory deficiency. No significant change was detected in the expression of COX11 (Fig. 2B), a gene involved in copper delivery to Cox1p. Although shy1 mutants are rescued by copper supplementation to the media, overexpression of COX11 and other genes involved in mitochondrial copper metabolism such as COX17, COX19, SCO1 and COX23 neither produced any suppression of the shy1 respiratory defect nor enhanced the suppression by copper supplementation (data not shown).

The mechanism of suppression by HAP4 over-expression is mediated through the over-expression of nuclear encoded COX structural subunits
In contrast to COX assembly factors, all the nuclear genes coding for COX subunits except COX5b, are transcriptionally co-regulated by the Hap activation complex, which interacts with the Hap2/3/5p consensus CCAAT target sequence in their promoters (19) (Supplementary Material, Table S1). In our experiments, northern blot analyses showed that in shy1 mutants undergoing a diauxic shift, overexpression of HAP4 induced a 1.7–1.9-fold increase in the steady-state levels of COX4, COX5a and COX6 mRNA (Fig. 2B). Overexpression of either COX4 or COX6 did not produce any significant effect on the ability of shy1 mutants to grow (Fig. 4A) and respire on EG (Fig. 4B). COX5a overexpression, however, increased shy1 cell respiration on minimum ethanol/glycerol media from 15–21% of wild-type values (Fig. 4B), resulting in a clearly observable growth of the mutant on this medium (Fig. 4A).


Figure 4
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Suppresion of shy1 by HAP4 overexpression is mediated by overexpression of Cox5ap and Cox6p. (A) The respiratory competent wild-type strain W303 and a strain carrying a null allele of shy1 ({Delta}shy1) transformed with either empty plasmids (ep) or plasmids over-expressing COX4, COX5a and COX6 alone or in combination were grown over-night in liquid selective WO-D media. Ten-fold serial dilutions of the two strains were plated on solid YPD or WO-EG media and incubated at 30°C. Pictures were taken after 2 and 5 days of incubation, respectively. (B) KCN-sensitive endogenous cell respiration was measured polarographically in the same strains than in (A) growing in WO-EG. The bars indicate the mean ± SD from at least three independent sets of measurements. (C) Diagram representing the strategy used to replace the promoter of COX5a by several mutated versions of the constitutive HAP4-independent transcription factor 1 (TEF) promoter retaining different levels of expression induction with respect to the unmutated TEF1 promoter (modified from 23). (D) The respiratory competent wild-type strain W303 and a strain carrying a null allele of shy1 ({Delta}shy1) overexpressing HAP4, either maintaining their endogenous promoter in COX5a or having it substituted by the mutated version of TEF1, TEF1-1, were grown over-night in liquid selective WO-D media. Ten-fold serial dilutions of the two strains were plated on solid YPD or WO-EG media and incubated at 30°C. Pictures were taken after 2 and 5 days of incubation, respectively. (E) Steady-state levels of COX5a mRNA detected by northern blot in the strains mentioned in (D).

 
Cox5ap is stabilized by Cox6p but not Cox4p (21). In addition, Cox5ap and Cox6p have been found to physically interact forming a sub-complex that accumulates in some COX assembly mutants (3). For these reasons, we decided to explore the effects of different combinations of the COX nuclear structural genes on respiration of shy1 mutants. Over-expression of COX4 in combination with each of the other genes did not produce any effect. However, over-expression of COX5a with COX6 significantly improved the growth on non-fermentable minimal ethanol/glycerol and increased the respiratory activity of the {Delta}shy1 mutant from 15 to 30–32% of wild type.

The enhanced efficiency of COX assembled in the absence of Shy1p can be explained by an increase in a subassembly containing Cox5ap and Cox6p. In these conditions a larger amount of newly synthesized Cox1p is spared from proteolysis, as observed in our chase experiments (Fig. 3B), and can be matured to form a ternary complex with Cox5ap–6p. This result is consistent with results obtained with human fibroblasts from LS patients carrying mutations in SURF1, which were found to accumulate a COX assembly intermediate consisting of subunit I and subunits CoxIV and CoxVa, the latter two being the human homologues of yeast Cox5ap and Cox4p, respectively (2). Hap4p increases cell respiration to 42% of wild-type, while concomitant over-expression of Cox5ap and Cox6p achieves a rate of 30% of wild-type, suggesting that other COX related factors may contribute to suppression by HAP4. In this line, it was recently reported that co-over-expression of COX10 enhanced the suppressor effect of MSS51 over-expression in a {Delta}shy1 strain, probably by increasing the amount of matured heme A-containing Cox1p (22).

To further test if suppression by HAP4 is mediated through higher expression of Cox5a and 6p, we changed the promoter of COX5a by inserting a constitutive HAP4-independent promoter using a yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 (translation and elongation factor 1) promoter (23). The promoter activities of these mutants range from 8–120% of the normal TEF1 promoter, independent of the carbon source. A cassette with promoter TEF1-1, which retain 100% of TEF1 activity, was used to replace the promoter of COX5a in wild-type and {Delta}shy1 strains overexpressing HAP4 (Fig. 4C). The substitution did not compromise the ability of the wild-type to grow in non-fermentable carbon sources (Fig. 4D), indicating that the amount of COX5a mRNA expressed was enough to sustain a wild-type respiration level. In the {Delta}shy1 mutant over-expressing HAP4, the substitution of the COX5a by the TEF1-1 promoter reduced the levels of COX5a mRNA to that of the {Delta}shy1 strain (Fig. 4E) and abolished the suppressed phenotype (Fig. 4D).

Effect of over-expression on shy1 mutants of the human CCAAT binding transcription factor NF-Y subunit A, homologue of yeast HAP2
The Hap complex is conserved from yeast to humans where the complex, termed NF-Y, contains three subunits, NF-YA, B and C, homologues of Hap2p, 3p and 5p, respectively. Some of the subunits are interchangeable across species. For example, human NF-YA, that contains both a DNA binding domain and a Q-rich activation domain, complements the respiratory defect of a hap2 yeast mutant (13). In our system, overexpression of human NF-YA was not able to suppress the {Delta}shy1 respiratory growth defect in YPEG but it did slightly increase its respiratory capacity from 15 to 20% similar to what was observed by overexpressing yeast HAP2 (Supplementary Material, Fig. S6).

Restoration of the COX assembly defect of fibroblasts from an LS patient carrying a mutation in SURF1 by over-expression of NF-YA
The Hap2/3/4/5 complex is the main activator of transcription of yeast nuclear genes involved in mitochondrial biogenesis and oxidative phosphorylation (19). In contrast, the mammalian genes are regulated by a variety of transcription factors, most notably NRF-1 and NRF-2 (24). Recently expression of at least two key proteins of the mammalian mitochondrial translational machinery, mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase, were shown to be regulated by NF-Y (25). Since NF-Y regulates expression of fatty acid oxidation genes, its stimulatory effect on mitochondrial biogenesis could be indirect as reported for other transcriptional factors such as the peroxisome proliferator-activated receptor PPAR{delta} in skeletal muscle (26).

We tested the effect NF-Y on a fibroblast cell line from an LS patient carrying a homozygous mutation in SURF1 (27) and displaying a severe COX deficiency, a phenotype that was also maintained after immortalization as demonstrated by the absence of the characteristic cytochemical staining (Supplementary Material, Fig. S7). Immunostaining for COX subunit 1 with an antibody conjugated to Alexa Fluor 488 showed green staining super-imposable on the mitochondrial network that was intense in normal fibroblasts but significantly weaker and more diffused in SURF1 mutant fibroblasts (Supplementary Material, Fig. S8). The difference in immunostaining is even more evident when wild-type and SURF1 mutant fibroblasts were mixed prior to staining (Supplementary Material, Fig. S8).

Fibroblasts from the SURF1-LS patient were transformed with an empty pIRES2 plasmid, and the same plasmid overexpressing either SURF1 of NF-YA. Cytochemical staining for COX activity 48 h after transfection showed that while the empty plasmid transfection failed to show an increase in COX activity, the fibroblasts over-expressing SURF1 (~10–12% of the culture) recovered COX activity (Fig. 5A). This was also true of the fibroblasts that had been transfected with NF-YA (~12–15% of the culture). The cultures from the SURF1 mutant patient transfected with either wild-type SURF1 or NF-YA showed cells with an intense COX I green signal that co-localized with the branched mitochondrial network stained with Mitotracker red (Fig. 5B). The percentage of COX positive cells is in agreement with a control transfection with the plasmid pEGF-N1 (Clonthech, Palo Alto, CA) expressing enhanced green fluorescent protein (eGFP), which on average yielded 15% cells were transfected expressing eGFP.


Figure 5
View larger version (51K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. COX expression in SURF1 fibroblasts overexpressing NF-YA. (A) Cytochemical cytochrome c oxidase activity staining. Wild-type (WT) and SURF1 mutant (surf1) fibroblasts were grown in cover-slips. The SURF1 mutant fibroblasts were transformed with an empty plasmid (pIRES2) or plasmids overexpressing either wild-type SURF1 or NF-YA. Forty eight hours after transfection, cells were submitted to cytochemical COX activity as described under the Materials and Methods. Pictures were taken at original magnification of 40x. (B) Fibroblast cultures were grown on coverslips, stained with Mitotracker red, fixed and immunochemically stained for COX subunit 1. Original magnification: 100x.

 
The restoration of COX by SURF1 and NF-YA was confirmed by assays of COX activity in cell homogenates obtained from the transfected cultures. Only ~16% of wild-type activity was detected in the SURF1 mutant fibroblasts. The COX activity measured in fibroblasts transfected with either wild-type SURF1 or NF-YA increased to ~25 or ~28% of wild-type, respectively, (Supplementary Material, Fig. S9) consistent with the values expected for full restoration of COX activity with a 15% transfection efficiency.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Shy1p/SURF1 is a conserved nuclear-encoded COX assembly factor that plays a role in maturation/assembly of subunit 1 of COX. Here we have reported that overexpression of the catalytic subunits of conserved CCAAT binding transcription activators suppress the COX assembly defect of both, yeast shy1 mutant cells and human SURF1 mutant fibroblasts.

The Hap2/3/4/5 transcriptional complex of yeast plays an important role in globally activating transcription of nuclear genes involved in mitochondrial respiration during transition from fermentation to respiration. This activation depends on the synthesis of the Hap4p subunit, which is regulated by carbon source (14). Our evidence indicates that over-expression of Hap4p rescues the COX assembly lesion and the respiratory defect of yeast shy1 mutants.

To identify the targets of Hap4p action responsible for the suppression, we first tested if suppression might be mediated through enhanced expression of MSS51, which was previously shown to suppress shy1 mutants. Expression of MSS51 was found to be independent of Hap4p thereby excluding its product from playing a role in the mechanism of suppression. This was also true of genes coding for proteins involved in heme A biosynthesis, mitochondrial copper homeostasis and other aspects of COX assembly. As an alternative approach, we over-expressed either singly or in combination candidate genes known to be regulated by the Hap complex.

This strategy revealed that over-expression of the nuclear-encoded Cox5ap subunit effectively compensated for absence of Shy1p. Suppression by COX5a was abolished when the normal promoter of this gene was replaced by a constitutive Hap-independent promoter, indicating that the mechanism of suppression depended minimally on Cox5ap over-expression. The more efficient rescue of the shy1 mutant transformed with COX5a in combination with COX6 but not COX4 indicated that suppression by Hap4p was also mediated by increased expression of Cox6p (Fig. 6A).


Figure 6
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Mechanisms of suppression of the COX assembly defect of yeast shy1 and human SURF1 mutants. (A) Different mechanisms of suppression of the yeast shy1 mutants. After Cox1p synthesis, this subunit is matured by insertion of heme and copper prosthetic groups, before or during the formation of an assembly intermediate containing Cox5ap and Cox6p. This ternary complex will subsequently assemble with metallated Cox2p. Shy1p could play a role either in Cox1p maturation or assembly with Cox2p. Mutant and additional copies of mss51 act as shy1 suppressors by increasing the amount of newly synthesized Cox1p, while overexpression of Hap4p suppresses mostly by increasing the amount of Cox5ap-Cox6p. (B) Model depicting the mechanism of suppression of the respiratory defect in yeast and human cellular models of Leigh's syndrome by overexpression of CCAAT binding transcription activators HAP4 and NF-YA, respectively.

 
COX assembly appears to be a sequential process (1,2) initiated by the synthesis of Cox1p, which during maturation of its metal and heme A prosthetic groups forms a subassembly with Cox5ap and Cox6p (mammalian CoxIV+CoxV) (2,3). These subunits are in direct contact with Cox1p in the mature enzyme (28). Cox5ap interacts with Cox1p through its single transmembrane domain, while Cox6p caps the matrix side of Cox1p. In S. cerevisiae, Cox1p is extremely prone to proteolysis in mutants blocked at any assembly step. Over-expression of Cox5ap and Cox6p may restore COX assembly by sparing newly synthesized Cox1p from proteolysis. Suppression of the shy1 mutant by HAP4 and MSS51sup is additive, presumably as a result of increased synthesis and availability of Cox1p for interaction with Cox5a and Cox6 and a higher flux through the assembly pathway (Fig. 6A).

Interestingly, the Cox1p–Cox5ap–Cox6p sub-complex accumulates in fibroblasts of SURF1 patients (2), although it does not accumulate in heme A deficient fibroblasts of patients with lesions in COX10 or COX15 (2,29). These findings suggested that the presence of heme A in Cox1p might stabilize its binding to Cox5ap and Cox6p. Because the two heme A groups are deeply buried within the interior of Cox1p (28) their insertion probably occurs either immediately after membrane insertion of the translated but unassembled subunit or during formation of the Cox1p–Cox5ap–Cox6p intermediate.

The presence of SURF1 orthologues in terminal oxidase operons of several prokaryotes (30) in which COX contains the evolutionary conserved core subunits (Cox1p, Cox2p, Cox3p) but not the supernumery subunits such as Cox5ap and Cox6p, suggests a role of Surf1p/Shy1p in forming the catalytic core of the enzyme. Studies in Rhodobacter sphaeroides, suggest that bacterial Surf1p is required either for insertion of heme A at the a3 center or stabilization of the a3-CuB binuclear center in Cox1p (6). A function of Shy1p in maturation of the a3 center would imply the existence of a second chaperone specific for the heme A of the cytochrome a center. Smith et al. (6) proposed that Surf1p could perform this role in an indirect way by interacting with subunits 1 or 2 to facilitate formation of the heme a3-CuB center. This would be in agreement with a previous observation suggesting an interaction of SURF1 with COXII in human cultured cells (31).

No interaction of yeast Shy1p was detected with either newly synthesized Cox1p or Cox2p (11). However, it is possible that such an interaction could occur only after the Cox1p–Cox5ap–Cox6p subassembly is formed and Cox2p has been matured by insertion of copper at the CuA site. The fact that SCO1- like SURF1-deficient fibroblasts accumulate the COXI–COXIV–COXVa (yeast Cox1p–Cox5ap–Cox6p) subassembly (2) strongly supports this possibility as SCO1 is required for the formation of CuA (32) and hence maturation of COXII.

Cox2p may associate with Cox1p–Cox5ap–Cox6p upon Cox2p metallation which in turn could affect the stability of the CuB-hem a3 center by capping the heme-insertion channel formed in Cox1p–Cox5ap-Cox6p subassembly (33). Shy1p could play a role in facilitating this interaction. Our results showing suppression of shy1 by increasing the amount of Cox1p–5a–6p supports this possibility although a direct role of Shy1p on Cox1p maturation cannot be excluded (Fig. 6A). Supporting a role of Shy1p as assembly factor, it was recently reported that it promotes COX biogenesis through association with different protein modules, potential COX assembly intermediates containing Cox1p and Cox5ap among other proteins (34).

Surf1p does not play a direct role in copper insertion into COX (6). However, we have observed that copper supplementation to the media is able to partially restore the respiratory defect of yeast shy1 mutants. In yeast shy1 mutants copper supplementation could facilitate the formation of the CuB site in a slightly larger pool of Cox1p that could proceed in the assembly process finally resulting in increased levels of the Cox1p–Cox5ap–Cox6p intermediate. Alternatively or maybe concurrently, copper supplementation could increase the pool of CuA containing Cox2p, which also would increase the pace of the assembly process in the absence of Shy1p. However, the suppressor effect was not enhanced by overexpression of mitochondrial copper metabolism genes including COX17, SCO1 and COX11. It is also intriguing that the suppressor effect of copper supplementation is not additive to the effect of neither increased amount of Cox1p by mutations in mss51 nor increased amounts of Cox5ap and Cox6p by overexpression of HAP4. In fact, copper supplementation is some how deleterious to the shy1 revertant strain with mutated mss51. If the amount of heme A available for Cox1p maturation in shy1 mutant is limiting, an excess of copper could contribute to build unstable pro-oxidant intermediates, like the ones recently proposed (35), when the amount of newly synthesized Cox1p is enhanced.

NF-Y regulates the expression of many genes involved in cell cycle and has also been proposed to play a role in transcriptional regulation of cellular aging through p53 (36). NF-Y and the transcriptional factor Sp1 (37) cooperate to regulate the expression of several genes involved in lipid metabolism including fatty acid synthase and carnitine palmitoyltransferase (CPT)-I. These genes are also regulated by peroxisome proliferator-activated receptor PPAR, a ligand-activated nuclear receptor that induces transcription of genes encoding fatty acid oxidation enzymes as well as the uncoupling proteins (26). An increase in PPAR{delta} activity induces increased muscle mitochondrial biogenesis (38). Because it has been recently reported that a high-fat diet induces increased mitochondrial biogenesis in muscle from rats through activation of the PPAR{delta} (39), it is possible that over-expression of NF-Y also produces a similar effect on mitochondrial biogenesis. Although mammalian NF-Y complex does not play a general role in expression of genes involved in mitochondrial biogenesis, it was recently shown to regulate the expression of two genes required for mitochondrial translation (25).

These reports prompted us to examine if over-expression of NF-Y in human SURF mutants fibroblasts could mimic the suppressor effect of HAP4 in yeast shy1 mutants. We showed that NF-YA over-expression is able to increase the mitochondrial COX in SURF1 deficient fibroblast. The mechanism of COX-deficiency suppression by NF-Y may occur by transcriptional activation of the same or a different set of genes than those proposed for suppression of yeast shy1 mutants by HAP4 (Fig. 6B). Knowledge of such mechanisms can be helpful in future attempts to devise therapeutic interventions to combat LS as well as other forms of mitochondrial COX deficiency.

In the wider context, our results would suggest that differences in the expression of oxidative phosphorylation (OXPHOS) structuraland assembly factor genes can modify disease mechanisms and may provide a basis for the influence of genetic background and variable tissue involvement in mitochondrial disorders. A greater understanding of the influence of genetic factors such as single nucleotide polymorphisms (SNPs) in OXPHOS-related genes and physiological changes in gene expression may therefore provide a better understanding of differences in the progression of inherited mitochondrial diseases between individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Yeast strains and media
Most of the work was carried out with the W303-1A wild-type strain (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) the isogenic mutant strain {Delta}shy1 in which the whole gene had been deleted by insertion of a URA3 marker (5) and the isogenic strain W125 carrying a point mutation in the region of SHY1 coding for the second transmembrane domain of Shy1p (9). A shy1 mutant with a point mutation in the SHY1 region coding for the first transmembrane domain of Shy1p (C173/U) was also used in some studies (9). The genotypes and source of these and other strains of S. cerevisiae strains are listed in Supplementary Material, Table S2. The compositions of the growth media have been described elsewhere (40) and are detailed in the Supplementary Materials and Methods.

Characterization of yeast mitochondrial respiratory chain
Mitochondria were prepared from strains grown in media containing 2% galactose, according to the method of Faye et al. (41) except that zymolyase 20T (ICN Biochemicals Inc., Aurora, OH) instead of Glusulase was used for the conversion of cells to spheroplasts.

Mitochondria prepared from the different strains were assayed polarographically for KCN-sensitive NADH oxidase and ascorbate -N-N-N’-N’-tetramathyl-p-phenilenediamine (TMPD) oxidation using a Clark type polarographic oxygen electrode from Hansatech Instruments (Norfolk, UK) at 24°C as described (5). Endogenous cell respiration was also assayed in the presence of galactose or ethanol–glycerol. The specific activities reported were corrected for KCN-insensitive respiration.

Mitochondria were also used for spectrophotometric assays carried at 24°C to measure KCN-sensitive cytochrome c oxidase (COX) activity and antimycin A-sensitive NADH cytochrome c reductase (NCCR) activity as described (5). Total mitochondrial cytochromes spectra was obtained as reported (42).

For in vivo analysis of mitochondrial protein synthesis, mitochondrial gene products were labeled with 35S-methionine in whole cells at 30°C in the presence of cycloheximide (5). Details of these and other methods are described in the Supplementary Materials and Methods.

Human cell lines, culture conditions, transfections, enzyme assays and immunodetections
A previously reported primary fibroblast line from an LS patient was obtained from Agnes Rötig (Necker Hospital, Paris, France) (27). The patient carried a homozygous 1-bp deletion within exon 6 of the SURF1 gene that causes a frameshift mutation resulting in a premature stop-codon at amino acid residue 187 of the polypeptide. The fibroblast cell lines were immortalized by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes. Human neonatal fibroblasts (CCD-10645 k) were obtained from ATCC (Manassas, VA) and used as a control cell line. Cells were cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/ml sodium pyruvate and 0.25 mg/ml uridine. For transient expression studies, the genes of interest were cloned into the plasmid pIRES2 (Clontech, Mountain View, CA) and transfected into human cell lines by using Lipofectamine-2000 (Invitrogen, Carlsbad, CA).

For immunocytochemistry, cells were grown on coverslips. Forty eight hours after transfection, living cells were incubated 20 min with 50 nM mitochondrial dye Mitotracker red (CMXRos; Molecular Probes, Invitrogen). Coverslips were washed with PBS and fixed with 2% paraformaldehyde in PBS, permeabilized with cold methanol and then incubated with an anti-COXI monoclonal antibody conjugated to Alexa Fluor 488 (Molecular Probes, Invitrogen) at a concentration of 2 µg/ml in 5% BSA in PBS for 2 h. For cytochemistry, cells were grown on coverslips. Forty eight hours after transfection, cells were stained for succinate dehydrogenase and cytochrome c oxidase activities as described (8).

To measure COX activity in cells homogenates, cells were collected by trypsinization 48 h after transfection, and submitted to three freezing–thawing cycles. COX activity was measure in the presence of lauryl maltoside as described (43).

Miscellaneous procedures
Standard procedures used for DNA cloning, bacterial and yeast transformation, northern and western blot analyses are described in the Supplementary Materials and Methods.

Statistical analysis
Most experiments were done at least in triplicate. Data are presented as means ± SD of absolute values or percent of control. The values obtained for wild-type, mutant and over-expressor strains for the different parameters studied were compared by Student' t-test. P < 0.05 was considered significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This research was supported by National Institutes of Health Research Grant GM071775A (to A.B.), GM50187 (to A.T.) and a Research Grant from the Muscular Dystrophy Association (to A.B.). F.F. is supported by Telethon-Italy, Fellowship no. GFP05008.


   ACKNOWLEDGEMENTS
 
We thank Dr C. Moraes, Dr S. Williams, D. Horn and I.C. Soto for critically reading the manuscript. We thank Dr A. Rötig (Hopital des Enfants Malades, Paris, France) for providing with the SURF1 deficient fibroblast cell line, Dr L. Pon (Columbia University, NY) for the anti MOM45 Ab, Dr C. Moraes (University of Miami, FL) for the HPV 16 E6/E7 oncogenes, Dr G. Stephanopoulos (Massachusetts Institute of Technology, Cambridge, MA) for the TEF1-based promoter replacement cassettes and Dr B. Grimpe (University of Miami, FL) for the pIRES2 plasmid.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

  1. Nijtmans L.G., Taanman J.W., Muijsers A.O., Speijer D., Van den Bogert C. Assembly of cytochrome-c oxidase in cultured human cells. Eur. J. Biochem. (1998) 254:389–394.[Web of Science][Medline]

  2. Williams S.L., Valnot I., Rustin P., Taanman J.W. Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1, or SURF1. J. Biol. Chem. (2004) 279:7462–7469.[Abstract/Free Full Text]

  3. Church C., Goehring B., Forsha D., Wazny P., Poyton R.O. A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant. J. Biol. Chem. (2005) 280:1854–1863.[Abstract/Free Full Text]

  4. Horan S., Bourges I., Taanman J.W., Meunier B. Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast. Biochem J. (2005) 390:703–708.[Web of Science][Medline]

  5. Barrientos A., Korr D., Tzagoloff A. Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome. EMBO J. (2002) 21:43–52.[CrossRef][Web of Science][Medline]

  6. Smith D., Gray J., Mitchell L., Antholine W.E., Hosler J.P. Assembly of cytochrome-c oxidase in the absence of assembly protein Surf1p leads to loss of the active site heme. J. Biol. Chem. (2005) 280:17652–17656.[Abstract/Free Full Text]

  7. Zhu Z., Yao J., Johns T., Fu K., De Bie I., Macmillan C., Cuthbert A.P., Newbold R.F., Wang J., Chevrette M., et al. SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet. (1998) 20:337–343.[CrossRef][Web of Science][Medline]

  8. Tiranti V., Hoertnagel K., Carrozzo R., Galimberti C., Munaro M., Granatiero M., Zelante L., Gasparini P., Marzella R., Rocchi M., et al. Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency. Am. J. Hum. Genet. (1998) 63:1609–1621.[CrossRef][Web of Science][Medline]

  9. Mashkevich G., Repetto B., Glerum D.M., Jin C., Tzagoloff A. SHY1, the yeast homolog of the mammalian SURF-1 gene, encodes a mitochondrial protein required for respiration. J. Biol. Chem. (1997) 272:14356–14364.[Abstract/Free Full Text]

  10. Tiranti V., Galimberti C., Nijtmans L., Bovolenta S., Perini M.P., Zeviani M. Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions. Hum. Mol. Genet. (1999) 8:2533–2540.[Abstract/Free Full Text]

  11. Barrientos A., Zambrano A., Tzagoloff A. Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J. (2004) 23:3472–3482.[CrossRef][Web of Science][Medline]

  12. Dell'agnello C., Leo S., Agostino A., Szabadkai G., Tiveron C., Zulian A., Prelle A., Roubertoux P., Rizzuto R., Zeviani M. Increased longevity and refractoriness to Ca2+-dependent neurodegeneration in Surf1 knockout mice. Hum. Mol. Genet. (2007) 16:431–444.[Abstract/Free Full Text]

  13. Becker D.M., Fikes J.D., Guarente L. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc. Natl Acad. Sci. USA (1991) 88:1968–1972.[Abstract/Free Full Text]

  14. Forsburg S.L., Guarente L. Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev. (1989) 3:1166–1178.[Abstract/Free Full Text]

  15. Steidl S., Tuncher A., Goda H., Guder C., Papadopoulou N., Kobayashi T., Tsukagoshi N., Kato M., Brakhage A.A. A single subunit of a heterotrimeric CCAAT-binding complex carries a nuclear localization signal: piggy back transport of the pre-assembled complex to the nucleus. J. Mol. Biol. (2004) 342:515–524.[CrossRef][Medline]

  16. McNabb D.S., Pinto I. Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA complex in Saccharomyces cerevisiae. Eukaryot. Cell (2005) 4:1829–1839.[Abstract/Free Full Text]

  17. Lascaris R., Bussemaker H.J., Boorsma A., Piper M., van der Spek H., Grivell L., Blom J. Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state. Genome Biol. (2003) 4:R3.[CrossRef][Medline]

  18. DeRisi J.L., Iyer V.R., Brown P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science (1997) 278:680–686.[Abstract/Free Full Text]

  19. Buschlen l., Amillet J.-M., Guiard B., Fournier A., Marcireau C., Bolotin-Fukuhara M. The S. cerevisiae HAP complex, a key regulator of mitochondrial function, cooordinates nuclear and mitochondrial gene expression. Comp. Funct. Genom. (2003) 4:37–46.[CrossRef]

  20. Schlaepfer I.R., Mattoon J.R., Bajszar G. The sequence and potential regulatory elements of the HEM2 promoter of Saccharomyces cerevisiae. Yeast (1994) 10:227–229.[CrossRef][Medline]

  21. Glerum D.M., Tzagoloff A. Submitochondrial distributions and stabilities of subunits 4, 5 and 6 of yeast cytochrome oxidase in assembly defective mutants. FEBS Lett. (1997) 412:410–414.[CrossRef][Web of Science][Medline]

  22. Pierrel F., Bestwick M.L., Cobine P.A., Khalimonchuk O., Cricco J.A., Winge D.R. Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly. EMBO J. (2007) 26:4335–4346.[CrossRef][Medline]

  23. Nevoigt E., Kohnke J., Fischer C.R., Alper H., Stahl U., Stephanopoulos G. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl. Environ. Microbiol. (2006) 72:5266–5273.[Abstract/Free Full Text]

  24. Scarpulla R.C. Nuclear control of respiratory gene expression in mammalian cells. J. Cell Biochem. (2006) 97:673–683.[CrossRef][Web of Science][Medline]

  25. Zanotto E., Shah Z.H., Jacobs H.T. The bidirectional promoter of two genes for the mitochondrial translational apparatus in mouse is regulated by an array of CCAAT boxes interacting with the transcription factor NF-Y. Nucleic Acids Res. (2007) 35:664–677.[Abstract/Free Full Text]

  26. Evans R.M., Barish G.D., Wang Y.X. PPARs and the complex journey to obesity. Nat. Med. (2004) 10:355–361.[CrossRef][Web of Science][Medline]

  27. von Kleist-Retzow J.C., Vial E., Chantrel-Groussard K., Rotig A., Munnich A., Rustin P., Taanman J.W. Biochemical, genetic and immunoblot analyses of 17 patients with an isolated cytochrome c oxidase deficiency. Biochim. Biophys. Acta (1999) 1455:35–44.[Medline]

  28. Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawa-Itoh K., Nakashima R., Yaono R., Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science (1996) 272:1136–1144.[Abstract]

  29. Antonicka H., Mattman A., Carlson C.G., Glerum D.M., Hoffbuhr K.C., Leary S.C., Kennaway N.G., Shoubridge E.A. Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy. Am. J. Hum. Genet. (2003) 72:101–114.[CrossRef][Web of Science][Medline]

  30. Poyau A., Buchet K., Godinot C. Sequence conservation from human to prokaryotes of Surf1, a protein involved in cytochrome c oxidase assembly, deficient in Leigh syndrome. FEBS Lett. (1999) 462:416–420.[CrossRef][Medline]

  31. Nijtmans L.G., Artal Sanz M., Bucko M., Farhoud M.H., Feenstra M., Hakkaart G.A., Zeviani M., Grivell L.A. Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast. FEBS Lett. (2001) 498:46–51.[CrossRef][Web of Science][Medline]

  32. Leary S.C., Kaufman B.A., Pellecchia G., Guercin G.H., Mattman A., Jaksch M., Shoubridge E.A. Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase. Hum. Mol. Genet. (2004) 13:1839–1848.[Abstract/Free Full Text]

  33. Cobine P.A., Pierrel F., Winge D.R. Copper trafficking to the mitochondrion and assembly of copper metalloenzymes. Biochim. Biophys. Acta (2006) 1763:759–772.[Medline]

  34. Mick D.U., Wagner K., van der Laan M., Frazier A.E., Perschil I., Pawlas M., Meyer H.E., Warscheid B., Rehling P. Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly. EMBO J. (2007) 26:4347–4358.[CrossRef][Medline]

  35. Khalimonchuk O., Bird A., Winge D.R. Evidence for a pro-oxidant intermediate in the assembly of cytochrome oxidase. J. Biol. Chem. (2007) 282:17442–17449.[Abstract/Free Full Text]

  36. Matuoka K., Chen K.Y. Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y. Ageing Res. Rev. (2002) 1:639–651.[CrossRef][Medline]

  37. Roder K., Wolf S.S., Larkin K.J., Schweizer M. Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1. Gene (1999) 234:61–69.[CrossRef][Medline]

  38. Tanaka T., Yamamoto J., Iwasaki S., Asaba H., Hamura H., Ikeda Y., Watanabe M., Magoori K., Ioka R.X., Tachibana K., et al. Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc. Natl Acad. Sci. USA (2003) 100:15924–15929.[Abstract/Free Full Text]

  39. Garcia-Roves P., Huss J.M., Han D.H., Hancock C.R., Iglesias-Gutierrez E., Chen M., Holloszy J.O. Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle. Proc. Natl Acad. Sci. USA (2007) 104:10709–10713.[Abstract/Free Full Text]

  40. Myers A.M., Pape L.K., Tzagoloff A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. (1985) 4:2087–2092.[Web of Science][Medline]

  41. Faye G., Kujawa C., Fukuhara H. Physical and genetic organization of petite and grande yeast mitochondrial DNA. IV. In vivo transcription products of mitochondrial DNA and localization of 23 S ribosomal RNA in petite mutants of Saccharomyces cerevisiae. J. Mol. Biol. (1974) 88:185–203.[CrossRef][Medline]

  42. Tzagoloff A., Akai A., Needleman R.B. Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes. J. Biol. Chem. (1975) 250:8228–8235.[Abstract/Free Full Text]

  43. Barrientos A., Kenyon L., Moraes C.T. Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency. J. Biol. Chem. (1998) 273:14210–14217.[Abstract/Free Full Text]

  44. Hlavacek O., Bourens M., Salone V., Lachacinski N., Lemaire C., Dujardin G. The transcriptional activator HAP4 is a high copy suppressor of an oxal yeast mutation. Gene (2005) 354:53–57.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
Y. Benita, H. Kikuchi, A. D. Smith, M. Q. Zhang, D. C. Chung, and R. J. Xavier
An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia
Nucleic Acids Res., August 1, 2009; 37(14): 4587 - 4602.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
L. Stiburek, K. Vesela, H. Hansikova, H. Hulkova, and J. Zeman
Loss of function of Sco1 and its interaction with cytochrome c oxidase
Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1218 - C1226.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Wang, Y. Wang, and E. L. Hegg
Regulation of the Heme A Biosynthetic Pathway: DIFFERENTIAL REGULATION OF HEME A SYNTHASE AND HEME O SYNTHASE IN SACCHAROMYCES CEREVISIAE
J. Biol. Chem., January 9, 2009; 284(2): 839 - 847.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
17/6/775    most recent
ddm349v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Fontanesi, F.
Right arrow Articles by Barrientos, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fontanesi, F.
Right arrow Articles by Barrientos, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?