Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion

Abstract

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to tRNA by the aa-tRNA synthetases. An exception is Gln-tRNA synthesis, which in eukaryotes is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ the transamidation pathway, in which a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts. Early work suggested that mitochondria use the transamidation pathway for Gln-tRNA formation. We found no biochemical support for this in Saccharomyces cerevisiae mitochondria, but demonstrated the presence of the cytoplasmic GlnRS in the organelle and its involvement in mitochondrial Gln-tRNA synthesis. In addition, we showed in vivo localization of cytoplasmic tRNAGln in mitochondria and demonstrated its role in mitochondrial translation. We furthermore reconstituted in vitro cytoplasmic tRNAGln import into mitochondria by a novel mechanism. This tRNA import mechanism expands our knowledge of RNA trafficking in the eukaryotic cell. These findings change our view of the evolution of organellar protein synthesis.

Figure 1.

Pathways to Gln-tRNA formation. The direct aminoacylation pathway (top) and the transamidation pathway (bottom) are both abundant in nature. GluAdT refers to the tRNA-dependent amidotransferase of the transamidation pathway.

Figure 2.

Aminoacylation of S. cerevisiae mitochondrial tRNAs. (A) Pure tRNA_m^Glu (○) and tRNA_m^Gln (▴) in the presence of S. cerevisiae mGluRS and [¹⁴C]-glutamate compared to tRNA_m^Gln (▪) in the presence of S. cerevisiae GlnRS and [¹⁴C]-glutamine; relative to minus enzyme control (⋄). (B) TLC analysis of [¹⁴C]-aminoacyl-tRNAs (aa-tRNAs) formed by the enzymes as indicated. [¹⁴C]-amino acids are compared to standards (std). (C) Total unfractionated yeast tRNAs were probed with [³²P]-labeled oligonucleotides specific for tRNA_m^Gln and tRNA_n^Gln as indicated. The intensity of the two tRNA bands reflects the very different abundances of the cytoplasmic and mitochondrial tRNA^Gln.(D) Acid urea gel analysis of aa-tRNAs extracted from total mitochondria (lane 1) and from in vitro aminoacylation reactions (lanes 2,3). Total mitochondrial tRNA was deacylated under basic condition (lane 2) and reaminoacylated with glutamine in vitro by GlnRS (lane 3). Aminoacylated mitochondrial tRNA Gln-tRNA_n^Gln (a) and deacylated tRNA_n^Gln (b) was visualized by Northern blot.

Figure 3.

Localization of tRNA_n^Gln to yeast mitochondria. (A) Northern analysis of total, cytosolic, and mitochondrial RNA fractions from wild-type yeast W303. RNAs were visualized with probes specific for tRNA_n^Gln, tRNA_m^Gln, and U6 snRNA. (B) RT-PCR analysis of total mitochondrial (Mito) and cytosolic (Cyto) RNA. Reactions with DNase-treated, total mitochondrial RNA (lane 2) or cytosolic RNA (lane 4), or in the absence of reverse transcriptase (RT) to control for contaminating DNA (lanes 3,5), were compared to a standard (lane 1). (C) Sequencing results of cloned RT-PCR products from lanes 2 and 4. Fiftyfive mitochondrial clones and 32 cytoplasmic clones were examined. The total number of each tRNA^Gln clones identified in the respective RNA pools is indicated. Representative chromatograms of RT-PCR clones are shown for tRNA_n^Gln_UUG1, tRNA_n^Gln_UUG2, and tRNA_n^Gln_CUG. The anticodon and position 42 are underlined.

Figure 4.

S. cerevisiae glutamine tRNAs. tRNA sequences are aligned under the predicted secondary structure (top line) where dots indicate unpaired bases and paired bases are indicated with a < or > (Lowe and Eddy 1997). Nuclear tRNA_n^Gln sequences, their chromosomal locations and anticodon designations are indicated (left), are compared to the single tRNA_m^Gln encoded in the mitochondrial genome. Conserved bases are shaded in gray. The anti-codon and position 42 are in bold with unique base changes shaded black to highlight differences between the tRNAs.

Figure 5.

In vitro import of native tRNA_n^Gln into isolated yeast mitochondria. (A) Nuclease protection of native tRNA_n^Gln incubated with isolated yeast mitochondria. Native tRNA_n^Gln radioactively labeled at the 5′ end with γ³²P-ATP (1 × 10⁵ cpm) was incubated with isolated yeast mitochondria in the presence (lane 1) and absence (lane 4) of ATP and subsequently digested with micrococcal nuclease (MN). The arrow depicts the migration of the full-length RNA protected from MN digestion. (Lane 2) As a control, input RNA was digested with MN in the absence of mitochondria. Lane 3 represents one-tenth of the input RNA, without MN digestion and in the absence of mitochondria, used for quantification. (B) In vitro import of native S. cerevisiae tRNA_n^Gln compared to S. cerevisiae tRNA_n^Lys and the Leishmania spliced leader RNA (slRNA) into isolated yeast mitochondria. Import experiments are presented as in A with the third lane in each panel representing an RNA-only control (one-tenth of the input RNA) used for quantification.

Figure 6.

Localization of yeast GlnRS to the mitochondrion. (A) GFP fluorescence in living cells shows GlnRS-GFP is colocalized in the cytoplasm and the mitochondria. DAPI staining of mitochondria can be overlapped with the mitochondrial GlnRS-GFP fluorescence. (B) Immunofluorescence shows a V5-GlnRS fusion protein is also colocalized to the cytosol and the mitochondria of a GlnRS deletion strain. Mitochondrial structures are visualized by coexpression of a COXIV-GFP fusion protein, which is localized only to the mitochondrion (Sesaki and Jensen 1999). (C) Western blot analysis of fractions from wild-type yeast W303: (C) 40 μg total cytosolic proteins; (M) 40 μg mitochondria; (MP_P) 80 μg mitoplast pellet; (MP_S) 40 μg mitoplast supernatant. GlnRS was visualized by an anti-GlnRS antibody and compared with marker antibodies for APRT (cytoplasm) and mtHsp70 (mitochondrial matrix).

Figure 7.

A single suppressor tRNA is active in both cytoplasmic and mitochondrial translation. (A) The yeast strain W303 (ura3-52 and trp1-1^UAG) containing an empty p316(URA3⁺) vector (EV) or p316 with an amber suppressor mutant tRNA_n^Gln_CUG gene (tRNA_n^Gln_CUA) was grown on media lacking uracil (-URA) or tryptophan (-Trp). tRNA_n^Gln_CUA-dependent suppression of the trp1-1^UAG mutation is shown by the ability of W303 to grow in the absence of tryptophan. (B) Cox2p expression in wild-type yeast (WT) or cox2-114^UAG (HM4) (Kolesnikova et al. 2000) visualized via immunofluorescence with an anti-Cox2p antibody. tRNA_n^Gln_CUA-dependent suppression of the cox2-114^UAG mutation is shown by the increased levels of Cox2p staining in HM4 cells containing the suppressor tRNA. Cells were costained with Mito Tracker.

Figure 8.

Origins of GlnRS from the eukaryotic cytoplasm. GlnRS, acquired by horizontal gene transfer, has replaced Glu-tRNA amidotransferase (GluAdT) in some bacteria and is of eukaryotic origin. In the mitochondrion, the GlnRS has replaced the bacterial-type GluAdT, which is part of a more ancient pathway.