In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function
2002; Springer Nature; Volume: 21; Issue: 24 Linguagem: Inglês
10.1093/emboj/cdf690
ISSN1460-2075
Autores Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoArticle16 December 2002free access In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function Antoine Margeot Antoine Margeot Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Corinne Blugeon Corinne Blugeon Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Julien Sylvestre Julien Sylvestre Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Stéphane Vialette Stéphane Vialette Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Claude Jacq Claude Jacq Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Marisol Corral-Debrinski Corresponding Author Marisol Corral-Debrinski Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Antoine Margeot Antoine Margeot Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Corinne Blugeon Corinne Blugeon Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Julien Sylvestre Julien Sylvestre Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Stéphane Vialette Stéphane Vialette Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Claude Jacq Claude Jacq Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Marisol Corral-Debrinski Corresponding Author Marisol Corral-Debrinski Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France Search for more papers by this author Author Information Antoine Margeot1, Corinne Blugeon1, Julien Sylvestre1, Stéphane Vialette1, Claude Jacq1 and Marisol Corral-Debrinski 1 1Laboratoire de Génétique Moléculaire, UMR CNRS 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:6893-6904https://doi.org/10.1093/emboj/cdf690 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We recently demonstrated that polysome-associated mRNAs that co-isolate with mitochondria encode a subset of mitochondrial proteins, and that the 3′ UTRs of these transcripts are essential for their localization to the vicinity of the organelle. To address the question of the involvement of the mRNA targeting process in mitochondrial biogenesis, we studied the role of ATP2 3′ UTR. An altered ATP2 allele in which the 3′ UTR was replaced by the ADH1 3′ UTR exhibits properties supporting the importance of mRNA localization to the vicinity of mitochondria: (i) the mutated strain presents a respiratory dysfunction; (ii) mito chondrial import of the protein translated from the altered gene is strongly reduced, even though the precursor is addressed to the organelle surface; (iii) systematic deletions of ATP2 3′ UTR revealed a 100 nucleotide element presenting RNA targeting properties. Additionally, when the ATM1 3′ UTR was replaced by the ADH1 3′ UTR, we obtained cells in which ATM1 mRNA is also delocalized, and presenting a respiratory dysfunction. This demonstrates that mRNA localization to the vicinity of mitochondria plays a critical role in organelle biogenesis. Introduction The biogenesis of mitochondria is a complex cellular process that involves the concerted expression of both nuclear and mitochondrial genomes. It is assumed that mitochondria contain ∼15–20% of cellular proteins (Kumar et al., 2002) and must import several hundreds of polypeptides encoded by the nuclear DNA (Pfanner and Geissler, 2001). Mitochondrial function is essential for the life of mammalian cells, as has been well documented for over 15 years in man (Wallace, 1999). The correct sorting of mitochondrial proteins is the first step to ensure organelle functionality. A classic mechanism for segregating mitochondrial proteins relies on targeting sequences that are either cleavable extensions or within the mature protein. The signal sequences are decoded by a dynamic, multisubunit transport machinery, the TOM and TIM complexes, which directs them to their correct destination within one of the four mitochondrial subcompartments: the outer membrane, the intermembrane space, the inner membrane and the matrix (Pfanner and Geissler, 2001). Alternatively, mounting evidence suggests that the localization of mRNAs to the vicinity of mitochondria is another mechanism employed by cells for mitochondrial protein sorting. In this case, a co-translational phase might assist the import of some precursors (Corral-Debrinski et al., 1999,2000; Fünfschilling and Rospert, 1999; Gratzer et al., 2000; George et al., 2002). Using yeast DNA microarrays to analyze the mRNA populations associated with free and mitochondrion-bound polysomes, we recently demonstrated that >100 mRNAs encoding mitochondrial proteins localize to mitochondrion-bound polysomes. These mRNAs have 3′ untranslated regions (3′ UTRs) conferring mitochondrion-targeting properties (Marc et al., 2002). We then decided to determine whether the mitochondrial localization of one of these mRNAs is essential for the function of the corresponding protein. ATP2 mRNA localizes exclusively to mitochondrion-bound polysomes, and encodes the β-subunit of the F1-ATP synthase or respiratory chain complex V. The use of an RNA-labeling system to visualize RNA localization in living cells (Beach et al., 1999; Corral-Debrinski et al., 2000) allowed us to demonstrate that the 3′ UTR of ATP2 contains sufficient information to address a reporter RNA to the vicinity of mitochondria. The replacement in vivo, by homologous recombination, of ATP2 3′ UTR with the 3′ UTR of ADH1, a gene encoding a cytoplasmic protein, leads to a respiratory-deficient strain. In these cells, not only is the altered ATP2 mRNA enriched in free cytoplasmic polysomes, but also the Atp2 protein is accumulated as a precursor poorly translocated into the organelle, even though its reaches the outer mitochondrial membrane. The significant decrease in the amount of mature Atp2p inside the mitochondria is responsible for the respiratory deficiency observed, as complementation with a wild-type form of ATP2 completely restores cell respiration. Therefore, ATP2 mRNA delivery to the vicinity of mitochondria is essential for the functionality of the respiratory chain. In an attempt to generalize the functional importance of 3′ UTRs, we replaced the 3′ UTR of ATM1, a gene coding for an ABC transporter of the inner mitochondrial membrane (Leighton and Schatz, 1995), by the ADH1 3′ UTR and we obtained a cell respiration impairment. In these cells, the altered ATM1 mRNA is over-represented in free cytoplasmic polysomes and the ability to grow on glycerol is restored by transformation with a wild-type ATM1 gene. Thus, the absence of ATM1 or ATP2 3′ UTRs leads to an abnormal cellular distribution of the corresponding transcripts associated with a respiratory deficiency, confirming that mRNA sorting to the vicinity of mitochondria represents a key step to ensure organelle function. Results Either the mts or the 3′ UTR of ATP2 is able to address a reporter RNA to the vicinity of mitochondria in living yeast cells The use of a whole-genome approach to identify mRNAs localized to the vicinity of mitochondria allowed a MLR (mitochondrial localization of mRNA) value of 98 out of 100 to be attributed to the ATP2 transcript, meaning that it localizes to mitochondrion-bound polysomes (Marc et al., 2002). We then decided to visualize RNA in living cells (Beach et al., 1999; Corral-Debrinski et al., 2000) to examine two sequences within ATP2 as possible regulators of RNA sorting: the mitochondrial targeting sequence (mts) and the 3′ UTR. The nucleotide region corresponding to the first 35 amino acids of Atp2p was fused to two CP (coat protein from the MS2 bacteriophage) binding sites in the pIIIA/MS2-2 plasmid, which will direct the synthesis of the reporter RNA. This RNA can be visualized by fluorescence microscopy when cells express the CP–GFP fusion protein. It has been well described in yeast that mitochondria consist of a branched tubular network, the continuity of which depends on a balanced frequency of fusion and fission events (Egner et al., 2002); furthermore, the size and the shape of the mitochondrial compartment depend on the carbon source (Stevens, 1977; Miyakawa et al., 1984). When cells were grown on galactose, we observed small discrete fluorescent spots, which represent mitochondria, as visualized with rhodamine B, a well-characterized mitochondrial probe (Johnson et al., 1980; Chen, 1989). Additionally, reporter RNA localized to mitochondrial nucleoids observed with the Hoechst reagent (Figure 1A). When cells were grown on the non-fermentable source glycerol, the reporter RNA was visualized in branched and more elaborated networks also stained by rhodamine B and Hoechst reagents. Therefore, the region coding for the mts of Atp2 is sufficient to address an RNA to the periphery of mitochondria, as we showed previously for the ATM1 mts (Corral-Debrinski et al., 2000). A region of 639 nucleotides (nt) between the ATP2 stop codon and the CAF17 AUG (CAF17 is contiguous to ATP2 in chromosome X) was inserted in the pIIIA/MS2-2 plasmid. The cellular distribution of this RNA was followed in cells expressing the CP–GFP fusion protein (Figure 1A). Hybrid RNA in these cells was present as small discrete fluorescent spots, which remarkably co-localized with Hoechst staining, representing mitochondrial nucleoids (Miyakawa et al., 1984) When cells were grown on glycerol, we observed the reporter RNA in the typical reticulum structure of mitochondria as visualized with rhodamine B (Figure 1A). When either the mts or the 3′ UTR was placed in the opposite orientation such that the non-coding sequence was transcribed, the reporter RNAs were diffuse in their distribution throughout the cytoplasm (not shown). Thus, information within the mts or the 3′ UTR of ATP2 is sufficient to promote export of the RNA from the nucleus and targeting to the periphery of mitochondria. To map more precisely cis-acting elements within the 639 nt of ATP2 3′ UTR, the distribution of various deletion RNAs was analyzed. Three consensus polyadenylation signals (AAUAAA) are present in this region: the first is situated 5 nt downstream of the stop codon, the second 239 nt farther and the third 302 nt downstream of the stop codon. The hybrid RNA containing the last 396 nt did not localize to the vicinity of mitochondria, and a diffuse staining in the cytoplasm was consistently observed, while the first 243 nt of the 3′ UTR are as efficient as the complete 3′ UTR, allowing the reporter RNA to produce a punctate distribution of fluorescent speckles that localize to mitochondrial structures (Figure 1B). The MFOLD program for RNA secondary structure (Zuker, 1989) predicted that the 250 nucleotide element could fold into a long stem–loop structure formed by four stems separated by asymmetric bulges (Figure 1C). To define further the minimal structural motif in this stem–loop, we examined the targeting properties of the region encompassing the first 150 nt. A reporter RNA with the region 50–150, in which the stem–loop structure was also predicted, presents the proper localization in close contact with mitochondria (Figure 1B). However, the reporter RNA encompassing the 1–100 region showed a diffuse cytoplasmic staining, suggesting that the reporter RNA does not localize to the vicinity of mitochondria (Figure 1B). Interestingly, the folding of this fragment does not predict any particular stable stem–loop structure (Figure 1C). These results demonstrate that specific regions within ATP2 3′ UTR play a crucial role in the mRNA targeting process. Figure 1.Imaging of fluorescent RNA in living yeast cells. The RNA-labeling system (Beach et al., 1999) was used to determine the addressing information included in both the 3′ UTR of the ATP2 gene and the sequence encoding the first 35 amino acids of Atp2p representing its mitochondrial targeting sequence (MTS). Co-expression of CP–GFP plasmid and reporter RNAs leads to formation of a GFP-labeled RNA, which was visualized using fluorescence microscopy techniques. (A) The ATP2 3′ UTR of 639 bp long or the sequence corresponding to Atp2p's mts of 105 bp long were cloned in the pIIIA/MS2-2 plasmid. Cells had grown either in 2% galactose or 2% glycerol medium and were visualized at early log phase. GFP indicates the green RNA labeling, H the Hoechst staining and R the rhodamine B labeling; cells were also photographed with Nomarski optics (N). (B) Several lengths of the ATP2 3′ UTR were inserted into the pIIIA/MS2-2 plasmid. Cells expressing each reporter RNA and the CP–GFP protein were grown in 2% galactose medium and visualized after Hoechst staining (H). Green RNA labeling is indicated (GFP) and cells were also photographed with Nomarski optics (N). (C) The MFOLD program for RNA secondary structure (Zuker, 1989) was applied to predict the stem–loop structures that can be formed in the different ATP2-3′UTR fragments tested in the RNA-labeling system. The arrows indicate the stable 70-nt-long stem–loop structure. Download figure Download PowerPoint In vivo substitution of the ATP2 3′ UTR by the ADH1 3′ UTR leads to respiratory-deficient cells associated with an abnormal localization of the ATP2 mRNA The localization of ATP2 mRNA to the vicinity of mitochondria seems to be dependent on at least two regions within the transcript: the mts and the 3′ UTR, as we have previously reported for ATM1 (Corral-Debrinski et al., 2000). We can predict that the in vivo modification of either of these elements could alter the final destination of the corresponding transcript. The 3′ UTR of ATP2 possesses the same targeting properties as the ATM1 3′ UTR (Figure 1) and is certainly involved in the localization of ATP2 mRNA to the vicinity of the mitochondria (Corral-Debrinski et al., 2000; Marc et al., 2002). In order to assess the biological role of the ATP2 mRNA targeting process, we modified the corresponding chromosomal allele by homologous recombination. Heterozygous diploids of the BMA64 strain were transformed with a PCR product of the pFA6a-3HA-TRP1 plasmid (Longtine et al., 1998), which was designed to recombine within the 3′ region of the chromosomal ATP2 allele. After dissection of each tetrad, two TRP+ spores were obtained, and were called ATP2-3′UTRADH1 cells. In these haploid cells, the C-terminus of the ATP2 ORF is fused to three tandem repeats of the influenza virus hemagglutin epitope (3HA), followed by the 3′ UTR of ADH1, a gene coding for a cytoplasmic protein (Figure 2A). These cells are unable to form colonies on media containing the non-fermentable carbon source glycerol at both 28 and 37°C. They also have a reduced growth rate in minimum synthetic medium (Figure 2B). The ability to grow on liquid glycerol medium was measured in wild-type and ATP2-3′UTRADH1 cells. As shown in Figure 2C, the growth rate of the mutated strains, MS1 and MS2, is dramatically reduced at both 28 and 37°C as compared with wild-type cells (WT). To exclude the possibility that the respiratory-deficient phenotype is due to the addition of the three HA epitopes at the C-terminus of Atp2p, we obtained BMA64 haploid cells, which express a HA-tagged Atp2 protein associated to the ATP2 3′ UTR. These cells, called B3 and B6, are able to grow on glycerol at both 28 and 37°C (Figure 2B and C); thus, the chromosomal ATP2 allele with a C-terminal triple HA tag functions like the wild-type allele. This result indicates that the respiratory chain deficiency of the ATP2-3′UTRADH1 strain is due to the absence of the ATP2 3′ UTR. Figure 2.In vivo substitution of the ATP2 3′ UTR by the ADH1 3′ UTR leads to a respiratory chain dysfunction. (A) The pFA6a-3HA1-TRP1 plasmid (Longtine et al., 1998) was used to obtain a modified strain (MS) from the diploid BMA64 strain called ATP2-3′UTRADH1 in which the C-terminus of ATP2 was fused to three tandem repeats of the HA epitope followed by the 3′ UTR of the ADH1 gene, encoding a cytoplasmic protein. After dissection of tetrads, each TRP+ spore was tested for its ability to grow on medium with the non-fermentable carbon source glycerol. (B) The ability to grow on glycerol plates was examined for two independent haploid TRP+ cells (M.S1, M.S2), for cells in which ATP2 locus was modified to obtain the synthesis of a protein with three tamdem repeats of the HA epitope while conserving its own 3′ UTR (B3, B6), and for wild-type BMA64 cells transformed with the pFL45 plasmid, allowing them to grow in the absence of tryptophan (WT). Cells were grown on complete synthetic medium containing 2% glucose and devoid of tryptophan to an OD of 2. They were serially diluted (1:5) and spotted either on complete synthetic medium devoid of tryptophan (CSM-Trp) or on 2% glycerol medium (Glycerol). The plates were then incubated for 2 days at both 28 and 37°C. (C) The growth rate of MS1 and MS2 cells was measured on liquid glycerol medium and compared with wild-type cells (WT) and with cells expressing the HA-tagged version of Atp2p (B3 and B6) either at 28 or 37°C. Cells were grown overnight on complete synthetic medium devoid of tryptophan and containing 2% glucose. The quantity of cells corresponding to an OD of 0.2 was diluted in 40 ml of glycerol medium. OD measurements were performed every 2 h. Download figure Download PowerPoint To determine whether this respiration dysfunction is the consequence of an abnormal localization of ATP2-3′UTRADH1 mRNA, fractionation experiments were performed to obtain free and mitochondrion-bound polysomes from wild-type and ATP2-3′UTRADH1 cells. RNAs were purified and subjected to northern blot analysis (Figure 3B). As expected, in wild-type cells, ATP2 and ATP3 transcripts (Marc et al., 2002) were remarkably enriched in mitochondrion-bound polysomes (Figure 3B, M-P). In ATP2-3′UTRADH1 cells, while ATP3 mRNA was enriched in mitochondrion-bound polysomes, the distribution of ATP2-3′UTRADH1 transcript was dramatically affected. Indeed, the mRNA predominantly localized to free cytoplasmic polysomes (Figure 3B, F-P), and very low amount of the overall ATP2 mRNA level was detected in mitochondrion-bound polysomes. The steady-state levels of ATP2 mRNA were similar in all the cells examined, as shown in northern blot experiments performed with total cellular RNA preparations (Figure 3A), indicating that the respiratory deficiency of the ATP2-3′UTRADH1 strain is not due to the instability of the RNA transcribed from the altered ATP2 allele. Therefore, the presence of ATP2 3′UTR is required for the accurate subcellular localization of the transcript in vivo. Figure 3.ATP2-3′UTRADH1 mRNA steady-state levels and subcellular localization in the ATP2-3′UTRADH1 strain. (A) Total RNAs were purified from ATP2-3′UTRADH1 cells (M.S1 and M.S2) and wild-type cells (WT), and subjected to northern blot analysis using successively ATP2 and ATP3 ORFs as radiolabeled probes. (B) Mitochondrion-bound polysomes (M-P) and free cytoplasmic polysomes (F-P) were purified from ATP2-3′UTRADH1 cells (M.S1 and M.S2) and wild-type cells (WT). Eight micrograms of RNA extracted from each polysomal population were separated on formaldehyde–agarose gels, subjected to northern blot analysis and hybridized successively with ATP2 and ATP3 probes. Autoradiograms shown in (A) and (B) represent an exposure time of 6 h at −80°C with Amersham intensifying screens. Methylene blue staining of the filters prior to hybridization is shown at the bottom. Download figure Download PowerPoint In the ATP2-3′UTRADH1 strain, Atp2 precursor is not efficiently translocated into the mitochondria To determine whether the Atp2 protein is produced at normal levels in the ATP2-3′UTRADH1 strain, we performed immunoblotting analyses using mitochondrial purifications. Two independent clones were examined; all the cells tested produced similar amounts of Atp2p. In ATP2-3′UTRADH1 cells (M.S1 and M.S2), very little of the mature form of the protein was detected: the majority of the fusion protein was present as the full-length precursor form of ∼62 kDa. In contrast, in wild-type cells (WT) almost all the Atp2p detected had an apparent mol. wt of ∼55 kDa, which corresponds to the mature form, in which the first 34 amino acids were cleaved (Bedwell et al., 1987) (Figure 4A). These data indicate that in the ATP2-3′UTRADH1 strain (M.S1), the mitochondrial import of Atp2p was impaired. To confirm the defect in Atp2p mitochondrial translocation, purified mitochondria from wild-type and ATP2-3′UTRADH1 cells were treated with proteinase K (PK) and subjected to western blot analyses (Figure 4B). The results for four independent experiments unambiguously showed that the mitochondrial import of Atp2p was significantly reduced in the ATP2-3′UTRADH1 strain. Nearly all the Atp2 protein signal in these cells was the full-length precursor form of ∼62 kDa. This protein was also detected with a specific anti-HA antibody, indicating that it is synthesized from the altered ATP2 allele (Figure 4B). Furthermore, we observed that in mitochondria purified from ATP2-3′UTRADH1 cells the Atp2 precursor was sensitive to proteolysis by PK, while in wild-type cells the majority of Atp2p was less accessible to proteolysis. Indeed, when PK concentrations used were >0.4 mg/ml, the protein produced from the altered ATP2 allele was almost entirely digested, whereas a significant amount of Atp2p produced in wild-type cells remained protease insensitive (Figure 4B), confirming that the majority of Atp2p produced from the altered allele is blocked on the surface of mitochondria. To examine the levels of other complex V proteins in the ATP2-3′UTRADH1 strain, western blots were performed using anti-Atp1p, anti-Atp4p and anti-Atp6p antibodies. The overall amounts of these proteins were not changed in the ATP2-3′UTRADH1 strain, suggesting that their mitochondrial import was not affected (Figure 4B). This also holds true for Abf2p, a mitochondrial DNA binding protein, the level of which was not affected in the mutated strain (Figure 4B). Additionally, we examined mitochondrial preparations of cells containing the chromosomal ATP2 allele with a C-terminal triple HA tag and the ATP2 3′UTR (B3 and B6). The fusion Atp2p is successfully translocated inside the mitochondria. Both the precursor and the mature form of the fusion protein were detected at the expected molecular weights when anti-Atp2p or anti-HA antibodies were used (Figure 4C), thus confirming that the addition of three HA epitopes at the C-terminus of Atp2p did not affect protein mitochondrial import, cleavage of the mts and assembly of the mature protein into complex V. This result is in agreement with the ability of these cells to grow on glycerol medium (Figure 2B and C). We can envisage that the modified form of ATP2 mRNA, which mainly localizes to free cytoplasmic polysomes, leads to the synthesis of a protein which, although recognized by the mitochondrial import machinery, is not successfully translocated into the organelle. Figure 4.Amount of Atp2 protein and localization in the ATP2-3′UTRADH1 strain. (A) Mitochondria were prepared from wild-type cells (WT) as from two independent clones of the ATP2-3′UTRADH1 strain (M.S1 and M.S2). SDS–PAGE was performed with 30 μg of proteins and analyzed by western blotting using antibodies against Atp2, Atp1 and Abf2 proteins.The precursor of Atp2p migrated at an approximate mol. wt of 62 kDa, while the mature protein migrated at ∼55 kDa. (B) To determine the precise cellular localization of Atp2p, mitochondria were treated for 30 min at 0°C with PK at 0.2, 0.4 or 0.6 mg/ml (PK); the PK digestion at 0.4 mg/ml was also performed in combination with 1% Triton X-100 (Triton). The following antibodies were used: polyclonal antibodies against Atp2p, Atp1p, Atp4p, Atp6p, Abf2p and monoclonal antibody 12CA5 against the HA epitope (second from the top). The precursor of Atp2p migrated at an approximate mol. wt of 62 kDa, while the mature protein migrated at ∼55 kDa. In wild-type mitochondria, no signal was detected with the 12CA5 antibody, thus confirming the identity of the revealed 62 kDa polypeptide with the protein expressed from the altered ATP2 allele. Moreover, the HA-tagged version of the Atp2 precursor was almost entirely digested by a 0.6 mg/ml concentration of PK, while the mature form of the protein in wild-type cells was more protected against digestion. These data indicate that Atp2 protein synthesized from the altered allele is present at the mitochondrial surface, but its mitochondrial import is not efficient. (C) To determine the cellular localization of a HA-tagged version of Atp2, mitochondria were purified from B3 and B6 cells and subjected to western blotting after digestion with 0.6 mg/ml PK in the presence or absence of 1% Triton X-100. As controls, CW04 strain (WT) and ATP2-3′UTRADH1 strain (MS2) were used. Antibodies against Atp2, Atp1 and Abf2 proteins and against the HA epitope (second from the top) were used successively. Even though we were able to detect more of the HA-tagged Atp2p precursor in B3 and B6 cells than in wild-type cells, the amount of the mature Atp2p insensitive to externally added protease in B3 and B6 cells was significantly increased as compared with that observed in the MS2 strain, thus confirming that the impairment of Atp2p mitochondrial import observed in the ATP2-3′UTRADH1 strain is not due to the presence of the three HA epitopes at the C-terminus of the protein. Download figure Download PowerPoint The expression of a wild-type version of ATP2 rescues the respiratory deficiency of the ATP2-3′UTRADH1 strain The above results establish a link between the respiratory deficiency and the decrease in Atp2p levels in the inner mitochondrial membrane of ATP2-3′UTRADH1 cells. To fully confirm the role of ATP2 mRNA localization in respiratory chain dysfunction, we transformed the modified strain with several low-copy-number plasmids containing the complete ATP2 ORF and different lengths of the ATP2 3′ UTR. A complete rescue of the respiratory capacity was obtained when ATP2-3′UTRADH1 cells were transformed with the pRSAM3 plasmid, which contains the full-length ATP2 3′ UTR. These cells are able to grow on glycerol medium at both 28 and 37°C as efficiently as wild-type cells (Figure 5A). The expression of pRSAM0 in the mutated strain led to a very low rate of growth on glycerol medium at 28°C, and these cells were unable to grow at 37°C. In this plasmid, the stop codon of Atp2p is followed by only 13 nt (Figure 5A). When the expression of ATP2 is directed from pRSAM1, which contains 150 nt of the ATP2 3′ UTR, cell growth on glycerol was improved at 28°C; however, the cell growth rate remained low at 37°C. The pRSAM2 encompassing 250 nt of the ATP2 3′ UTR allowed a full rescue of cell respiration; cells expressing ATP2 from this plasmid were able to respire at both 28 and 37°C, and their growth rate was identical to that observed in both wild-type cells and cells transformed with pRSAM3 (Figure 5A). Figure 5.Complementation experiments of the ATP2-3′UTRADH1 strain. (A) The ATP2-3′UTRADH1 strain (M.S1) was transformed with an array of low-copy-number plasmids expressing the ATP2 ORF in combination with different lengths of the ATP2 3′ UTR (pRSAM0-pRSAM3); as a control, the ATP2-3′UTRADH1 strain transformed with the empty pRS416 vector was used (M.S1). The ability of the transformed cells to grow on glycerol plates was determined at both 28 and 37°C. Cells were grown on complete synthetic medium containing 2% glucose and devoid of tryptophan to an OD of 2. They were serially diluted (1:5) and spotted either on complete synthetic medium devoid of tryptophan and containing 2% glucose (Glucose) or on 2% glycerol medium (Glycerol). The plates were then incubated for 2 days. Wild-type cells transformed with pRS416 and pFL45 (WT) were used as a positive control, since they are able to grow on glycerol and in the absence of tryptophan and uracile. (B) To compare the amount of Atp2p produced from each pRSAM plasmid, mitochondria from wild-type cells (WT) and transformed ATP2-3′UTRADH1 cells (M.S1) were analyzed by western blotting using antibodies against Atp2, Atp1 and Atp4 proteins. The precursor of Atp2p migrated at an approximate mol. wt of 62 kDa, while the mature protein migrated at the expected size of ∼55 kDa. (C) The table summarizes the size of the 3′ UTR in each tested plasmid, their ability to rescue cell respiration and the targeting properties determined in Figure 1. A clear correlation
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