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. 2002 Dec 16;21(24):6893-904.
doi: 10.1093/emboj/cdf690.

In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function

Antoine Margeot  1 , Corinne Blugeon, Julien Sylvestre, Stéphane Vialette, Claude Jacq, Marisol Corral-Debrinski
Affiliations

In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function

Antoine Margeot et al. EMBO J. .

Abstract

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) mitochondrial 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.

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Figures

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Fig. 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.
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Fig. 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.
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Fig. 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.
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Fig. 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.
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Fig. 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.
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Fig. 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 can be drawn between the length of the 3′ UTR, the level of the mature protein inside mitochondria (Figure 5B), the phenotypic complementation and the ability of the sequence to target a reporter RNA to the vicinity of mitochondria.
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Fig. 6. In vivo substitution of the ATM1 3′ UTR by the ADH1 3′ UTR also 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 ATM1-3′UTRADH1 in which the C-terminus of ATM1, the gene encoding an ABC transporter of the inner mitochondrial membrane (Leighton and Schatz, 1995), 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. Three tetrads are shown (1–3), all the Trp+ cells grew poorly on glycerol at 28°C. Two independent clones (MS.1 and MS.2) were transformed with a wild-type version of the ATM1 gene obtained by gap repair and their ability to grow on glycerol was compared with that of wild-type cells transformed with the pFL45 plasmid and with that of the original MS.1 and MS.2 cells. 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 (Glu-Trp) or on 2% glycerol medium (Glycerol). The plates were then incubated for 2 days at 28°C. (B) The growth rate of ATM1-3′UTRADH1 (MS.ATM1) cells was measured on liquid glycerol medium and compared with wild-type cells (WT), and with cells expressing the wild-type ATM1 gene (MS.ATM1+ATM1) and the ATP2-3′UTRADH1 strain (MS1.ATP2 and MS2.ATP2) 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. (C) To determine the subcellular localization of the modified ATM1 mRNA in the ATM1-3′UTRADH1 strain, mitochondrion-bound polysomes (M-P) and free cytoplasmic polysomes (F-P) were purified from ATM1-3′UTRADH1 cells (MS.ATM1) 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 ATM1 and ATP3 probes. Autoradiograms shown represent an exposure time of 16 h for ATM1 and of 6 h for ATP2 at –80°C with Amersham intensifying screens. Methylene blue staining of the filters prior hybridization is also shown.
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