![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 2, 523-535, February 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Department of Neurology and Biochemistry and Molecular Biology, The John T. Macdonald Foundation Center for Medical Genetics, University of Miami School of Medicine, Miami, FL 33136; Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703; and Department of Biological Sciences, Columbia University, New York, NY 10027
Submitted September 8, 2006;
Revised November 3, 2006;
Accepted November 20, 2006
Monitoring Editor: Peter Walter
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Tzagoloff and Dieckmann, 1990; Barrientos et al., 2002a; Solans et al., 2004).
In Saccharomyces cerevisiae, translation of each mitochondrial COX mRNA depends on one or more translational activator (for review, see Towpik, 2005). These mRNA-specific translational factors are either integral or peripheral inner membrane proteins that recognize the 5'-untranslated region (UTR) of their target transcripts. Current speculations suggest that translational activators may couple translation to cotranslational insertion of the newly synthesized hydrophobic products into the membrane near or at the site of their assembly into multisubunit complexes (Michaelis et al., 1991; Naithani et al., 2003). Most activators, Mss51p being an exception, interact with each other (Brown et al., 1994; Naithani et al., 2003), suggesting some level of coregulation in the expression of the core-forming subunits of COX (Fiori et al., 2005). Interactions have also been noted between the transcription factor Nam1p and translational activators, including Pet111p, Pet309p, and Pet494p (Naithani et al., 2003; Krause et al., 2004) raising the possibility that mitochondrial transcription may be coupled to translation.
COX1 is the mitochondrial gene for subunit1/Cox1p, an important catalytic subunit of COX containing copper and heme A prosthetic groups. Translation of COX1 in S. cerevisiae is under the control of the MSS51 and PET309 gene products that are also involved in maturation of the COX1 mRNAs (Decoster et al., 1990; Manthey and McEwen, 1995). Mutations in MSS51 or overexpression of the wild type gene can suppress shy1 null mutants by enhancing synthesis of Cox1p (Barrientos et al., 2002b). The function of Shy1p in maturation and/or assembly of Cox1p (Barrientos et al., 2002b; Perez-Martinez et al., 2003; Smith et al., 2005) is of considerable interest because mutations in its human homologue, Surf1p, are responsible for most diagnosed cases of Leigh's syndrome presenting a COX deficiency (Tiranti et al., 1998; Zhu et al., 1998).
Cox1p synthesis is suppressed in most COX assembly mutants, including shy1 mutants, but it is restored to normal levels by mss51 suppressors of shy1 or by mutations in COX14 (Barrientos et al., 2004), which codes for a COX assembly factor (Glerum et al., 1995). Like other translational activators Mss51p acts on the 5'-UTR to initiate translation (Perez-Martinez et al., 2003); however, mss51 mutants, unlike pet309 mutants, cannot be suppressed by changes in the 5'-UTR of COX1 mRNA. (Perez-Martinez et al., 2003; Barrientos et al., 2004). In addition, Mss51p acts on a target in the protein coding sequence of COX1 mRNA to promote elongation (Perez-Martinez et al., 2003). Mss51p and Cox1p form a transient complex (Perez-Martinez et al., 2003; Barrientos et al., 2004) that is stabilized by Cox14p (Barrientos et al., 2004). These interactions have been postulated to down-regulate Cox1p synthesis when COX assembly is impaired (Barrientos et al., 2004). According to this model, the release of Mss51p from the ternary complex and its availability for Cox1p synthesis occur at a downstream step in the assembly pathway, most likely catalyzed by Shy1p (Barrientos et al., 2004). Further studies are necessary to clarify the functional significance of the interactions of Mss51p on the 5'-UTR and the coding sequence of COX1 mRNA.
In the present study, we report a novel mitochondrial translation product, named mp15, with an apparent mass of 
15 kDa. This protein is detected in mss51 null mutants blocked in Cox1p synthesis and in strains carrying null alleles in nuclear genes coding for COX subunits or assembly factors in which the synthesis of Cox1p is reduced to 10% or less of wild type. We present evidence that mp15 is a truncated translation product of a partially unprocessed COX1 mRNA and that it is synthesized by an Mss51p- but not Pet309-independent mechanism. We also present evidence that binding of Mss51p to the 5'-UTR of COX1 mRNA is necessary for optimal initiation of translation by Pet309p, whereas the interaction of Mss51p with newly synthesized Cox1p may regulate elongation of the nascent polypeptide.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
|
). Equivalent amounts of total cellular proteins were separated by SDS-PAGE on a 17.5% polyacrylamide gel, transferred to a nitrocellulose membrane, and exposed to a Kodak X-OMAT x-ray film. Deviations from this procedure are described in some figure legends.
Construction of COX Mutant Strains Carrying a Proteolytically Inactive Version of yta10
The haploid strain W303
yta10/Yta10E559Q (Table 1) was obtained from Prof. T. Langer (Institut für Genetik und Zentrum für Molekulare Medizin, Universität zu Köln, Köln, Germany). This strain was crossed to a W303shy1 strain and to W303cox11. Diploid cells were sporulated and haploid progeny with the two parental mutant alleles (yta10 + shy1 or yta10 + cox11) and the yta10E559Q allele were isolated.
Protein Purification and Sequencing
Mss51p fused with the 26-kDa glutathione S-transferase (GST) was expressed from an integrative plasmid (pG96/ST13) in a strain carrying a null mutant allele of mss51 as reported previously (aW303mss51/ST13) (Barrientos et al., 2004). This strain was respiratory competent and grew on nonfermentable carbon sources with a doubling time similar to the parental wild type strain (Barrientos et al., 2004). Mitochondria were prepared from aW303mss51/ST13 strain by the method of Faye et al. (1974) except that zymolyase 20,000 (ICN Biochemicals, Aurora, OH) instead of glusulase was used to digest the cell wall. Mitochondrial proteins (5 mg) were solubilized with 1% lauryl maltoside in the presence of 0.5 M KCl and 1 mM phenylmethylsulfonyl fluoride. The extract was clarified by centrifugation at 50,000 x gav for 30 min and incubated with glutathione-Sepharose beads for 4 h at 4°C. After centrifugation at 1500 rpm for 5 min, the beads were washed three times with phosphate-buffered saline. The Mss51p–GST fusion protein was eluted with 10 mM glutathione, 50 mM Tris-base, pH 8.0, and concentrated using Vivaspin 500 columns. The concentrate was separated in a 12% SDS-PAGE system, transferred to a polyvinylidene fluoride membrane and stained with Coomassie blue. A prominent band of 68 kDa was excised from the membrane and used to sequence the N-terminal seven residues with a Procise Sequencer (Applied Biosystems, Foster City, CA) in the Proteomics facility of the University of Miami.
Yeast Three-Hybrid System (Y3H)
A Y3H system to map the interacting domain in Mss51p and its target in the 5'-UTR of COX1 mRNA was developed by using the commercially available RNA-Protein Hybrid Hunter kit (Invitrogen, Carlsbad, CA). Portions of the COX1 mRNA 5'-UTR were cloned into the plasmid pRH3' and portions, of Mss51p were expressed from the plasmid pYESTrp2. The host strain L40-ura-ms2 was cotransformed with pairs of pRH3'/and pYESTrp2/constructs, and the transformants were selected on yeast synthetic medium missing the appropriate auxotrophic markers (Ura– and Trp–) and histidine. The His–-deficient medium selected for expression of the HIS3 reporter gene, e.g., for colonies in which the RNA–protein interaction was produced. Inclusion of 5 mM 3-minotriazole, a competitive inhibitor of the HIS3 reporter protein, suppressed background growth in the His– medium. Cotransformations of the host strain with pair-combinations of the parent plasmids and one of the constructs served as controls. Interactions were confirmed by assaying for 
-galactoside, the second reporter using a filter lift assay as described previously (Breeden and Nasmyth, 1985).
Miscellaneous Procedures
Standard procedures were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from Escherichia coli (Sambrook et al., 1989). Yeast were transformed by the method of Schiestl and Gietz (1989). The one-step gene insertion method (Rothstein, 1983) was used to integrate linear plasmids at the URA3 or LEU2 locus of yeast nuclear DNA. Mitochondrial RNA was prepared from isolated mitochondria by modified extraction with hot-acidic phenol (Ausubel et al., 1994).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Manthey and McEwen, 1995). In agreement with earlier studies, mutations in either of these genes abolish synthesis of Cox1p (Figure 1A). However, a new low-molecular-weight polypeptide with a migration intermediate between Atp6p and Atp8p in SDS-PAGE was detected among the mitochondrial translation products labeled with [35S]methionine in the mss51 null mutant. Expression of wild-type MSS51 from integrative and episomal plasmids in this mss51 null mutant strain completely restored its ability of synthesizing Cox1p and abolished the synthesis of the new polypeptide (data not shown). This protein was absent in the pet309 mutant and in a pet309/mss51 double mutant (Figure 1A). The absence of the novel polypeptide in the pet309 mutant suggested that it could be a truncated product of COX1 requiring Pet309p for initiation of translation but destined for premature termination in the absence of Mss51p. To further establish the origin of the new polypeptide, mtDNA of M5-16/A3, a mutant with a large deletion in COX1 (Tzagoloff et al., 1975) was transferred by cytoduction to an mss51 null mutant and its parental wild type, both lacking mtDNA (
o strains). No synthesis of Cox1p or the novel protein was seen in the cox1 mutant either in the presence or absence of the mss51 null mutation (Figure 1B). These data constitute strong evidence that the novel protein, which we have named mp15, is encoded by a COX1 transcript.
|
The mp15 detected in mss51 mutants could be a novel translation product of a COX1 transcript or a degradation product of Cox1p. The fact that no Cox1p is detected in mss51 mutants, even after pulses of up to 60 min (data not shown), suggested that there is no synthesis of full-length Cox1p. Detection of mp15 after a 2.5-min pulse, the shortest time needed to observe a clear pattern of labeled proteins in the in vivo experiments (data not shown), argues against degradation of Cox1p as the source of mp15.
Radiolabeled mp15 separated by SDS-PAGE migrates slightly above 14-kDa marker (Figure 2) indicating a molecular weight of 15,000. Because hydrophobic proteins do not always separate true to size, the actual molecular weight could be larger.
|
Pet309-dependent Synthesis of mp15 Polypeptide Occurs in COX Assembly-arrested Mutants Displaying Reduced Synthesis of Cox1p
Mss51p was proposed to play a role in adjusting translation of Cox1p to the availability of its partners subunits during assembly of COX (Barrientos et al., 2002b, 2004). This was based on observations that synthesis of Cox1p is greatly reduced in most COX assembly mutants (Barrientos et al., 2004; Figure 3, A and B). The small amount of Cox1p detected in such mutants may reflect the fraction of this subunit that is associated with and stabilized by Mss51p and Cox14p (Perez-Martinez et al., 2003; Barrientos et al., 2004). Assembly-defective mutants in which Mss51p is tied up with unassembled Cox1p would be expected to have substantially reduced amounts of Mss51p available for new rounds of Cox1p translation, thereby allowing synthesis of mp15.
|
; Figure 3, A and B). Synthesis of mp15 was also abolished or greatly reduced in double mutants in which a null allele in a COX assembly gene was combined with a deletion of COX14 (Figure 3B). Such strains were previously shown to synthesize Cox1p normally (Barrientos et al., 2004).
Synthesis of mp15 in the mss51 Mutant Is Not Affected by a cox14 Mutation
Cox14p plays an important role in regulating translation of COX1 mRNA. In other studies, we have shown that cox14 mutants synthesize normal amounts of Cox1p. Moreover, the cox14 mutation cures the Cox1p translational defect of most COX mutants (Barrientos et al., 2004). This was interpreted to indicate that Cox14p may be necessary to stabilize the interaction of Mss51p with Cox1p. Accordingly, mutants lacking Cox14p would have more Mss51p available for translation of Cox1p.
The synthesis of mp15 is independent of Cox14p, because incorporation of [35S]methionine into mp15 was the same in the mss51 single mutant and in a strain with mutation in both mss51 and cox14 (Figure 4A). Overexpression of COX14 from the episomal plasmid pG93/T1 (Glerum et al., 1995) in a mss51 null mutant strain also did not influence the synthesis of mp15. These results support a role of Cox14p in regulating the availability of Mss51p for translation by trapping the protein in a ternary complex with full-length Cox1p, rather than acting to inhibit translational elongation.
|
), which was interpreted to be due to the accumulated larger pools of nuclear-encoded factors (i.e., Mss51p) required for mitochondrial gene expression. We wanted to test now if the enhanced synthesis of Cox1p in most COX assembly mutants had a modifying effect on the amount of newly synthesized mp15. We first tested the effect of CAP preincubation on mp15 synthesis in mss51 mutants. Incorporation of [35S]methionine into mp15 in an mss51 null mutant strain was substantially lower in CAP-pretreated cells (Figure 5, A and B). Although at variable levels, in some experiments, the amount of newly synthesized mitochondrial ribosomal protein Var1 was also reduced in mss51 mutants pretreated with CAP, suggesting an effect of the antibiotic on ribosome metabolism that could be also related to the reduced synthesis of mp15. However, the amount of newly synthesized mp15 was consistently reduced in CAP-pretreated mss51 mutant cells even in experiments in which the amount of newly synthesized Var1 was not affected. The reduction in mp15 was unlikely to be due to faster turnover, because it was observed at the shortest times of labeling. Pulse-chase experiments also showed that mp15 synthesized in 10 min (Figure 5, B and C) or 15 min of pulse (data not shown) with or without the CAP pretreatment were degraded at similar rates.
|
).
Mp15 Is a Membrane Protein and the Yta10p/Yta12p Protease Is Partially Responsible for Its Degradation
Cox1p is an integral protein, embedded in the mitochondrial inner membrane with 12 transmembrane 
-helices (Tsukihara et al., 1996). To test whether mp15 is also a membrane-bound protein, we have determined its solubility properties. Sonic irradiation of mss51 spheroplasts containing newly synthesized 35S-labeled mitochondrial translation products solubilized -ketoglutarate dehydrogenase, a soluble protein of the mitochondrial matrix, but not mp15 nor any of the other newly synthesized proteins that are known to be cotranslationally inserted in the membrane (Figure 6A). These results suggest that also mp15 is cotranslationally inserted in the mitochondrial inner membrane.
|
50% of the newly synthesized mp15 to be degraded after a 1-h chase (Figure 6B). The stability of unassembled Cox1p and of mp15 was also assessed in different COX mutants. In agreement with our previous findings, the small amount of labeled Cox1p present in the mutants was stable for at least 2 h of chase (Barrientos et al., 2004; Figure 6C). In contrast, mp15 was almost completely degraded after the 2-h chase.
Turnover of mitochondrially synthesized COX subunits that do not enter the assembly pathway is a function of different proteases, including the m-AAA protease complex Yta10p/Yta12p (Arlt et al., 1996), which also acts as a chaperone for assembly of the mitochondrial respiratory chain complexes (Arlt et al., 1996). The protease function can be inactivated by the E559Q mutation in the active site of Yta10p without affecting the chaperone function of the complex (Arlt et al., 1996). The role of the Yta10p/Yta12p in turnover of mp15 was studied in shy1/yta10 and a cox11/yta10 double mutant harboring the yta10E559Q allele. In vivo pulse-chase experiments of the double mutants revealed that mp15 is a substrate of Yta10p/Yta12p protease as a considerable amount of this novel protein was spared from degradation during the 1–2 h of chase in the background of the yta10E559Q allele (Figure 6C).
Synthesis of mp15 Is Affected by the Intron Composition of COX1
W303mss51, an mss51 null mutant containing W303 mtDNA, had significantly more mp15 than the same mutant with D273 mtDNA (Figure 1). The only significant difference in the COX1 genes of the two strains is the presence of additional introns in the gene of D273 mtDNA (Figure 7). In the absence of Mss51p, splicing of COX1 pre-mRNA containing a particular combination of introns may result in the accumulation of a particular splicing intermediate that serves as an mRNA for the aberrant mp15 translation product.
|
o derivatives of mss51 and shy1 null mutants. In vivo labeling of the mitochondrial products in the resultant strains showed that mp15 was detected as a prominent band in strains containing the mitochondrial genome of W303 but not D273 (Figure 1B). To confirm that the efficient production of mp15 was related to its COX1 gene, mtDNA from Wi04, a strain with the same COX1 introns as W303, was also transferred to the mss51 and shy1 null mutants. Synthesis of mp15 in both mutants was comparable with that seen in the same strains with the W303 mtDNA (data not shown). Based on the in vivo labeling results obtained with other mss51 mutants harboring different COX1 genes, however, it was not possible to relate the synthesis of mp15 to the absence of a particular intron, although the presence of aI1 and 5
seems to be an absolute requirement (Figure 7). No synthesis of mp15 was seen in C199, a strain with D273 mtDNA and a point mutation in mss51 causing partial loss of function (data not shown). This was also true of five other mss51 point mutants in the same mitochondrial genetic background (data not shown). Substitution of D273 by W303 mtDNA in C199, however, allowed some limited synthesis of mp15. These results could suggest that expression of mp15 is at least partially dependent on the nuclear background or that the synthesis of mp15 is reduced in leaky mutants of mss51.
mp15 Is Translated from a Partially Processed COX1 Transcript
Synthesis of mp15 occurs in strains lacking Mss51p but not Pet309p, and additionally, it requires a COX1 gene with a particular set of introns. Because processing and translation of COX1 transcripts are interdependent events, splicing of some COX1 introns being dependent on translation of intron-encoded maturases (Carignani et al., 1983, 1986), mp15 is likely to be derived from an incompletely processed COX1 transcript. It was not excluded, however, that mp15 might be translated from an aberrantly spliced or partially degraded COX1 transcript. To rule out these possibilities, we examined the COX1 transcripts by Northern blot analysis of total mitochondrial RNAs of mss51, pet309, and shy1 mutants (Figure 8). Even though the amount of mature COX1 mRNA was reduced in the mss51 mutant, as reported previously (Simon and Faye, 1984), the exon A5/intron aI5 probe revealed a similar pattern of COX1 precursors in the mss51 and shy1 mutants (Figure 8A). No new small mRNA species were found in either strain (Figure 8A). The probe against the entire coding sequence of COX1, confirmed the presence both mature and partially processed transcripts in the mss51 mutant with a COX1 gene containing four or more introns (W303) (Figure 8B). In agreement with previous reports, neither mature nor precursor COX1 RNAs were present in the pet309 mutant because of their instability in this genetic background (Manthey and McEwen, 1995). Mature COX1 mRNA was also detected in two point mutants of mss51 in the D273 background, C199 and C283 (data not shown).
|
Mss51p Binds to the 5'-UTR of COX1 mRNA
Mss51p has been conjectured to act on the COXI mRNA 5'-UTR to initiate translation and on the coding sequence to promote elongation (Decoster et al., 1990; Perez-Martinez et al., 2003). Because mp15 is only detected in the W303 and D273 mtDNA backgrounds, it is conceivable that the presence of a particular sequence(s) in the 5'-UTR of COX1 of these strains could by-pass the requirement of Mss51p for translation initiation but not elongation. Under these circumstances, synthesis of Cox1p could be prematurely terminated.
The putative interaction/s of Mss51p with the 5'-UTR of COX1 mRNA has not been characterized. We have used the Y3H depicted in Figure 9A to define both the RNA target(s) sequence and the Mss51p domain involved in the binding to the RNA. The constructs used as baits consisted of 460 base pairs 5'-UTR sequence (Manthey and McEwen, 1995) as well as different regions of the 5'-UTR with part of the first exon of the COX1 gene (Figure 9B). To ensure targeting of the Mss51p/Gal4 fusion proteins to the nucleus, a requirement in the Y3H system, the mitochondrial leader peptide of Mss51p was deleted. The mitochondrial import sequence of Mss51p determined by protein sequencing of Mss51p-GST purified from mitochondria consists of the N-terminal 35 residues encoded by the gene.
|
The region of the 5'-UTR of COX1 that was ascertained to interact with Mss51p did not display any nucleotide variation in the mtDNAs of D273, W303, and the strain with the intronless gene.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Together with Pet309p, it interacts with the 5'-UTR of the COX1 mRNA to initiate translation (Perez-Martinez et al., 2003). The yeast three-hybrid experiments reported here suggest that the target in the 5'-UTR of COX1 mRNA could be within 245 nucleotides upstream of the initiation codon. The sequence in Mss51p that could interact with this activation site has been mapped to a hydrophilic region located in the N-terminal 177 residues of the mature protein.
As noted previously, mss51 mutants lack Cox1p; unexpectedly, however, we found that they synthesize a novel 15-kDa protein. Several lines of evidence suggest that mp15 is translated from a COX1 transcript. This protein is not present in pet309 mutants in which initiation of COX1 translation is blocked and which do not accumulate mature or precursor COX1 mRNAs when this mitochondrial gene contains more than four introns (Manthey and McEwen, 1995). In addition, no mp15 is detected in an mss51/pet309 double mutant or in a mutant with a large deletion in COX1.
Translation of a Cox1p-related protein in mss51 mutants is at variance with previous observations that the interaction of Mss51p with the 5'-UTR of COX1 is essential for translation of Cox1p. This was evident by the failure of an mss51 mutant to express ARG8m when the recoded gene was substituted for COX1 and was fused to the normal 5'-UTR of COX1 (Perez-Martinez et al., 2003). The possibility that the requirement for Mss51p for translation is by-passed in mp15-producing strains as a result of a polymorphism in the COX1 5'-UTRs has been excluded as no sequence differences were found in their Mss51p-interacting domain. These observations suggest that translation of mp15 is initiated from an ATG other than the normal COX1 initiation codon. The dependence of mp15 expression on Pet309p, a protein implicated in translation initiation of COX1 mRNA (Manthey and McEwen, 1995), may be related to the already mentioned absence of COX1 transcripts in pet309 mutants and therefore does not exclude the use of an alternate start codon. Whether translation of mp15 starts in an exon or intron is unclear, although an exonic ATG is more likely for several reasons. The partially overlapping peptide patterns of Cox1p and mp15 indicate the presence in the latter of some exon-encoded sequences. Because the reading frames of COX1 introns are in register with the reading frames of their upstream but not downstream exons, a 15-kDa product could be expressed exclusively from an mRNA containing intron sequences but not from a reading frame initiated in an intron and encompassing the downstream exon.
Alternatively, mp15 could result from premature termination of translation of a COX1 transcript. Recent evidence has shown that Mss51p acts on target/s within the coding sequence of the COX1 mRNA or to the protein itself, to promote translational elongation and that in the absence of Mss51p, nascent Cox1p may block its own synthesis (Perez-Martinez et al., 2003). Translational regulation by nascent chains has been reported previously (for review, see Tenson and Ehrenberg, 2002).
The mp15 polypeptide is most evident in mss51 mutant containing mtDNA of W303, to a lesser extent in mss51 mutants with D273 mtDNA, and it is not detected in strains with an intronless COX1 or with combinations of introns other than those of W303 and D273. The appearance of novel mitochondrial translation products has been reported previously. For example, a cox1 mutant expressing both Pet309p and Mss51p but defective in intron aI1 splicing was shown to produce a 90-kDa protein translated from the continuous reading frame encoded by the first exon and part of the first intron of COX1. This protein is post-translationally cleaved into two polypeptides of 20 and 68 kDa, the latter constituting the maturase encoded by intron aI1 (Carignani et al., 1983). In contrast, synthesis of mp15 depends on the complete absence of Mss51p or presence of only limiting amounts of the translationally competent protein. That mp15 is not detected in an mss51 null mutant with intronless mtDNA supports the notion that this protein is translated from a COX1 precursor transcript with one or more introns. The COX1 genes of W303 and D273 contain all three group II introns (aI1, -2, and -5) and the group I intron aI3. The intron composition of COX1 in D273 is similar to W303 except for the presence of group I intron aI4, which seems to suppress mp15 synthesis. It is also noteworthy that the only difference between the COX1 genes of D273 and G1–224, which does not express mp15, is the absence in the latter of intron aI5. The lack of a clear correlation between the presence or absence of a particular intron and mp15 synthesis suggests that the overall intronic composition in the context of the mss51 mutation may be crucial for the production or stability of the transcript corresponding to the mp15 mRNA.
Cox1p synthesis is regulated by downstream events occurring during assembly of the COX holoenzyme (Perez-Martinez et al., 2003; Barrientos et al., 2004). In our model, entry of newly translated Cox1p into the COX assembly pathway depends on its interaction with Mss51p and Cox14p (Figure 10). When engaged in this ternary complex, Mss51p is forestalled from initiating new rounds of Cox1p synthesis. Dissociation of Mss51p from the complex is envisioned to occur when Cox1p acquires its prosthetic groups or interacts with other COX subunits. This step may be catalyzed by Shy1p (Perez-Martinez et al., 2003; Barrientos et al., 2004). This is supported by the severe repression of Cox1p translation in mutants that are blocked in COX assembly (Barrientos et al., 2004), in which mp15 is also synthesized because in these strains the amount of Mss51p available for normal COX1 mRNA translation is limited. The single exception are cox14 mutants, in which normal amounts of Cox1p are synthesized and Mss51p is available for translation even in the absence of COX assembly (Barrientos et al., 2004). For simplicity, our model depicted in Figure 10. does not include the role of Mss51p on the coding sequence of COX1 mRNA. As mentioned above, the interaction of Mss51p with Cox1p itself could be necessary for elongation of the nascent polypeptide and it could in addition regulate Cox1p synthesis (Perez-Martinez et al., 2003).
|
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Antoni Barrientos (abarrientos{at}med.miami.edu)
Abbreviations used: CAP, chloramphenicol; COX, cytochrome c oxidase; GST, glutathione S-transferase; UTR, untranslated region; Y3H, yeast three-hybrid system.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1994). Saccharomyces cerevisiae. In: Current Protocols in Molecular Biology, New York: Wiley, Vol. 2, 13.
Barrientos, A., Barros, M. H., Valnot, I., Rotig, A., Rustin, P., Tzagoloff, A. (2002a). Cytochrome oxidase in health and disease. Gene 286, 53–63.[CrossRef][Medline]
Barrientos, A., Korr, D., Tzagoloff, A. (2002b). Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome. EMBO J 21, 43–52.[CrossRef][Medline]
Barrientos, A., Zambrano, A., Tzagoloff, A. (2004). Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J 23, 3472–3482.[CrossRef][Medline]
Barros, M. H., Myers, A. M., Van Driesche, S., Tzagoloff, A. (2006). COX24 codes for a mitochondrial protein required for processing of the COX1 transcript. J. Biol. Chem 281, 3743–3751.
Barros, M. H., Nobrega, F. G., Tzagoloff, A. (2002). Mitochondrial ferredoxin is required for heme A synthesis in Saccharomyces cerevisiae. J. Biol. Chem 277, 9997–10002.
Breeden, L. and Nasmyth, K. (1985). Regulation of the yeast HO gene. Cold Spring Harb. Symp. Quant. Biol 50, 643–650.
Brown, N. G., Costanzo, M. C., Fox, T. D. (1994). Interactions among three proteins that specifically activate translation of the mitochondrial COX3 mRNA in Saccharomyces cerevisiae. Mol. Cell. Biol 14, 1045–1053.
Carignani, G., Groudinsky, O., Frezza, D., Schiavon, E., Bergantino, E., Slonimski, P. P. (1983). An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae. Cell 35, 733–742.[CrossRef][Medline]
Carignani, G., Netter, P., Bergantino, E., Robineau, S. (1986). Expression of the mitochondrial split gene coding for cytochrome oxidase subunit I in S. cerevisiae: RNA splicing pathway. Curr. Genet 11, 55–63.[CrossRef][Medline]
Carlson, C. G., Barrientos, A., Tzagoloff, A., Glerum, D. M. (2003). COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae. J. Biol. Chem 278, 3770–3775.
Conde, J. and Fink, G. R. (1976). A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc. Natl. Acad. Sci. USA 73, 3651–3655.
Decoster, E., Simon, M., Hatat, D., Faye, G. (1990). The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria. Mol. Gen. Genet 224, 111–118.[Medline]
Faye, G., Kujawa, C., Fukuhara, H. (1974). 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 88, 185–203.[CrossRef][Medline]
Feinberg, A. P. and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem 132, 6–13.[CrossRef][Medline]
Fiori, A., Perez-Martinez, X., Fox, T. D. (2005). Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly in mitochondria of Saccharomyces cerevisiae. Mol. Microbiol 56, 1689–1704.[Medline]
Glerum, D. M., Koerner, T. J., Tzagoloff, A. (1995). Cloning and characterization of COX14, whose product is required for assembly of yeast cytochrome oxidase. J. Biol. Chem 270, 15585–15590.
Glerum, D. M., Muroff, I., Jin, C., Tzagoloff, A. (1997). COX15 codes for a mitochondrial protein essential for the assembly of yeast cytochrome oxidase. J. Biol. Chem 272, 19088–19094.
Glerum, D. M., Shtanko, A., Tzagoloff, A. (1996a). Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase. J. Biol. Chem 271, 14504–14509.
Glerum, D. M., Shtanko, A., Tzagoloff, A. (1996b). SCO1 and SCO2 act as high copy suppressors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae. J. Biol. Chem 271, 20531–20535.
Glerum, D. M. and Tzagoloff, A. (1997). Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants. FEBS Lett 412, 410–414.[CrossRef][Medline]
Hell, K., Tzagoloff, A., Neupert, W., Stuart, R. A. (2000). Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2. J. Biol. Chem 275, 4571–4578.
Krause, K., Lopes de Souza, R., Roberts, D. G., Dieckmann, C. L. (2004). The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex. Mol. Biol. Cell 15, 2674–2683.
Labouesse, M. (1990). The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns. Mol. Gen. Genet 224, 209–221.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Manthey, G. M. and McEwen, J. E. (1995). The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae. EMBO J 14, 4031–4043.[Medline]
McEwen, J. E., Ko, C., Kloeckner-Gruissem, B., Poyton, R. O. (1986). Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups. J. Biol. Chem 261, 11872–11879.
Michaelis, U., Korte, A., Rodel, G. (1991). Association of cytochrome b translational activator proteins with the mitochondrial membrane: implications for cytochrome b expression in yeast. Mol. Gen. Genet 230, 177–185.[CrossRef][Medline]
Myers, A. M., Pape, L. K., Tzagoloff, A. (1985). Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J 4, 2087–2092.[Medline]
Naithani, S., Saracco, S. A., Butler, C. A., Fox, T. D. (2003). Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Mol. Biol. Cell 14, 324–333.
Nobrega, M. P., Bandeira, S. C., Beers, J., Tzagoloff, A. (2002). Characterization of COX19, a widely distributed gene required for expression of mitochondrial cytochrome oxidase. J. Biol. Chem 277, 40206–40211.
Nobrega, M. P., Nobrega, F. G., Tzagoloff, A. (1990). COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase. J. Biol. Chem 265, 14220–14226.
Perez-Martinez, X., Broadley, S. A., Fox, T. D. (2003). Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J 22, 5951–5961.[CrossRef][Medline]
Rothstein, R. J. (1983). One-step gene disruption in yeast. Methods Enzymol 101, 202–211.[Medline]
Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schagger, H. and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem 166, 368–379.[CrossRef][Medline]
Schiestl, R. H. and Gietz, R. D. (1989). High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet 16, 339–346.[CrossRef][Medline]
Seraphin, B., Boulet, A., Simon, M., Faye, G. (1987). Construction of a yeast strain devoid of mitochondrial introns and its use to screen nuclear genes involved in mitochondrial splicing. Proc. Natl. Acad. Sci. USA 84, 6810–6814.
Siep, M., van Oosterum, K., Neufeglise, H., van der Spek, H., Grivell, L. A. (2000). Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae. Curr. Genet 37, 213–220.[CrossRef][Medline]
Simon, M. and Faye, G. (1984). Steps in processing of the mitochondrial cytochrome oxidase subunit I pre-mRNA affected by a nuclear mutation in yeast. Proc. Natl. Acad. Sci. USA 81, 8–12.
Smith, D., Gray, J., Mitchell, L., Antholine, W. E., Hosler, J. P. (2005). Assembly of cytochrome-c oxidase in the absence of assembly protein Surf1p leads to loss of the active site heme. J. Biol. Chem 280, 17652–17656.
Solans, A., Zambrano, A., Barrientos, A. (2004). Cytochrome c oxidase deficiency: from yeast to human. Preclinica 2, 336–348.
Souza, R. L., Green-Willms, N. S., Fox, T. D., Tzagoloff, A., Nobrega, F. G. (2000). Cloning and characterization of COX18, a Saccharomyces cerevisiae PET gene required for the assembly of cytochrome oxidase. J. Biol. Chem 275, 14898–14902.
Tenson, T. and Ehrenberg, M. (2002). Regulatory nascent peptides in the ribosomal tunnel. Cell 108, 591–594.[CrossRef][Medline]
Tiranti, V., et al. (1998). Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency. Am. J. Hum. Genet 63, 1609–1621.[CrossRef][Medline]
Towpik, J. (2005). Regulation of mitochondrial translation in yeast. Cell. Mol. Biol. Lett 10, 571–594.[Medline]
Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yoshikawa, S. (1996). The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272, 1136–1144.[Abstract]
Tzagoloff, A., Akai, A., Foury, F. (1976). Assembly of the mitochondrial membrane system XVI. Modified form of the ATPase proteolipid in oligomycin-resistant mutants of Saccharomyces cerevisiae. FEBS Lett 65, 391–395.[CrossRef][Medline]
Tzagoloff, A., Akai, A., Needleman, R. B., Zulch, G. (1975). Assembly of the mitochondrial membrane system. Cytoplasmic mutants of Saccharomyces cerevisiae with lesions in enzymes of the respiratory chain and in the mitochondrial ATPase. J. Biol. Chem 250, 8236–8242.
Tzagoloff, A., Capitanio, N., Nobrega, M. P., Gatti, D. (1990). Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3. EMBO J 9, 2759–2764.
Tzagoloff, A. and Dieckmann, C. L. (1990). PET genes of Saccharomyces cerevisiae. Microbiol. Rev 54, 211–225.
Zhu, Z., et al. (1998). SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet 20, 337–343.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  O. Khalimonchuk, M. Bestwick, B. Meunier, T. C. Watts, and D. R. Winge Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase Mol. Cell. Biol., February 15, 2010; 30(4): 1004 - 1017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Fontanesi, I. C. Soto, D. Horn, and A. Barrientos Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis Mol. Cell. Biol., January 1, 2010; 30(1): 245 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Perez-Martinez, C. A. Butler, M. Shingu-Vazquez, and T. D. Fox Dual Functions of Mss51 Couple Synthesis of Cox1 to Assembly of Cytochrome c Oxidase in Saccharomyces cerevisiae Mitochondria Mol. Biol. Cell, October 15, 2009; 20(20): 4371 - 4380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
F. Pierrel, O. Khalimonchuk, P. A. Cobine, M. Bestwick, and D. R. Winge Coa2 Is an Assembly Factor for Yeast Cytochrome c Oxidase Biogenesis That Facilitates the Maturation of Cox1 Mol. Cell. Biol., August 15, 2008; 28(16): 4927 - 4939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bauerschmitt, S. Funes, and J. M. Herrmann The Membrane-bound GTPase Guf1 Promotes Mitochondrial Protein Synthesis under Suboptimal Conditions J. Biol. Chem., June 20, 2008; 283(25): 17139 - 17146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Khalimonchuk, A. Bird, and D. R. Winge Evidence for a Pro-oxidant Intermediate in the Assembly of Cytochrome Oxidase J. Biol. Chem., June 15, 2007; 282(24): 17442 - 17449. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
