Proposed mechanism of enolase involvement in tRK1 mitochondrial targeting. GIP indicates the general insertion pore, the outer membrane preprotein import complex composed of TOM proteins. Aminoacylated tRK1 is recognized by enolase (most probably Eno2p) in the cytoplasm and addressed toward the mitochondrial surface, where the tRNA is transferred to the peri-mitochondrially synthesized preMsk1p, while enolase inserts in the hypothetical glycolytic multiprotein complex, whose role may be the channelling of the pyruvate to the organelle. Further targeting of tRK1 to the mitochondrial matrix involves the functional GIP (Tarassov et al. 1995b).

Soluble pool of Eno2p is required for tRK1 import. (A) Effect of antibodies on tRK1 mitochondrial import. The antibodies used are indicated at the top. As 1× concentration, we used the concentration of antibodies with the same immunological capacity (determined by Western analysis). The left panel corresponds to tRK1 import into antibody pretreated mitochondria; the right panel corresponds to tRK1 import directed by immunodepleted IDP. (B) tRK1 import assays directed by IDP isolated from yeast strains indicated at the first line on the top (see explanations below the panel) into mitochondria isolated from different strains (second line). (Bottom) Efficiency of tRK1 import. (+Eno2p) Addition of 0.1 μg of recombinant Eno2p to the IDP of the strain indicated.

Translation of MSK1 mRNA is compartmentalized. Quantification results obtained after RT–qPCR measurement of mRNAs coding for Atp1 (first subunit of the mitochondrial ATP synthase), Krs1 (cytoplasmic lysyl–tRNA synthetase), Msk1 (preMsk1p), and Eno1/2 (enolase 1 and 2; oligonucleotide primers used do not distinguish between the two mRNAs). To obtain the indicated values, the absolute amounts of mRNAs were first calculated using purified T7-transcripts corresponding to each analyzed mRNA, then the ratio between each mRNA amount in the mitochondrially associated RNA pool and in the total RNA was calculated. In the control experiment, the isolation of mitochondria for RNA purification was performed in the absence of cycloheximide (−CYH).

Interaction of Eno2p with tRK1. (A) Detection of the interaction between Eno1/2p and tRK1 in vivo by the three-hybrid approach. The strategy is presented in the upper panel. MS2 RNA-fused tRK1 or tRK2 (nonimported RNA) were assayed for interaction with either Eno1p or Eno2p. Empty MS2 RNA gene-containing vector (MS2) and empty pGAD-GH plasmid (pGAD) were used as controls. To detect interaction, yeast L40-coat strain transfected cells were grown in the absence or presence of 10 mM 3AT, as indicated. (B–D) Analysis of RNA–protein interactions by gel-shift. Autoradiographs of native PAGE-separations of labeled tRK1 in the presence of preMsk1p (B) and/or Eno2p (C). Deacylated tRK1 [tRK1(da)] and aminoacylated tRK2 [tRK2(aa)] were used as controls of the specificity of interaction. Concentrations of the recombinant proteins in nM are indicated below the panels. (KMC) tRK1–preMsk1 complex; (KEC) tRK1–Eno2p complex; (tRK1) unbound labeled tRNA. (D) The effect of Eno2p presence on tRK1–preMsk1p complex formation.

Identification of import factors. (A) Strategy of proteins fractionation. (IDP) Protein extract. (Top and bottom right panels) In vitro import assays and autoradiographs of protected tRK1 after the incubation with isolated yeast mitochondria. (Input) An aliquot (1%) of labeled tRK1 used in each import assay. Ammonium sulphate saturation percentage and the numbers of mono-Q fractions are indicated. The middle panel represents results of Western immunodetection of preMsk1p in the same ammonium sulphate fractions as above. (B) North–Western analysis of the proteins from the mono-Q fraction 8 (Q8). The left panel is the Coomassie-stained gel, and the two panels on the right show autoradiographs after incubation of proteins with 5′-[³²P]-labeled tRK1 or tRK2. (C) In vitro tRK1 import assay with fraction Q8 proteins and recombinant Eno1p or Eno2p in the presence or absence of preMsk1p. [IDP(−M)] IDPs lacking preMsk1p. tRK1 import efficiencies are indicated below the autoradiograph. (D) Comparison of enzymatic and tRK1 import-directing activities of a normal (wild-type [WT]) and mutant recombinant versions of Eno2, containing the following substitutions: N207A, H159F, or H373F. On the left is the in vitro import assay in the presence of recombinant preMsk1p, with Eno2p versions indicated above; on the right is the quantification of import-directing efficiency and enolase enzymatic activity of the recombinant proteins.

A part of Eno2p colocalizes with mitochondria. (A) Western analyses of mitochondrially associated proteins. After the treatment indicated below the picture, the mitochondrial pellets or the total material were analyzed, as indicated. The antibodies used were Aco1p, aconite hydratase, 85 kDa, matrix (MM) localization; Tom70p, 70 kDa, outer membrane (MOM) associated; and CCH1HLp, Cytochrome c heme lyase, 30 kDa, inner membrane space (IMS) protein. Aco1p and CCH1HLp are known to be resistant to carbonate treatment (Diekert et al. 2001). (B,C) In vitro assay of mitochondrial localization using reticulocyte lysate-synthesized [³⁵S]-labeled proteins incubated with isolated organelles. (B) Autoradiographs of gel-fractionated proteins after treatment of mitochondria as indicated. (I) Input; (SW) swelling; (NaCl) treatment with 200 mM of salt; (Na₂CO₃) washing with 200 mM carbonate; (Triton X-100) treatment with 10% detergent; (P) pellet; (S) supernatant. (C) Autoradiograph of the gel-fractionated proteins after treatment of the mitochondria with 50 μg/mL of proteinase K (PK). Mitochondrially imported preMsk1p was used as a reference. (D) Analysis of Eno2p localization in yeast cells by confocal microscopy. Eno2p was YFP-labeled, mitochondria were localized by mito-tracker red. Zones of colocalization are indicated by white arrows on the merge image. (E) Assay for Eno1/2p interaction with immobilized phospholipids. (LPA) Lisophosphatidic acid; (LPC) lisophosphocholine; (PI) phosphatidyl-inositol; (PI3/4/5) phosphatidyl-inositol-3/4/5-phosphate; (PE) phosphatidylethanolamine; (PC) phosphatidylcholine; (S1P) sphingosine-1-phosphate; (PI3, 4/3,5/4,5P2) phosphatidyl-inositol-3,4/3,5/4,5-diphosphate; (PI3, 4,5P3) phosphatidyl-inositol-3,4,5-triphosphate; (PA) phosphatidic acid; (PS) phosphatidylserine.

Effect of ENO1/2 genes inactivation on tRK1 import and mitochondrial functions. (A) The effect of ENO1 and ENO2 genes deletion on yeast cells growth on nonfermentable carbon source (Glycerol [Gly] YPEG medium), YPGal medium (Gal), or YPD (Glu). Culture growing temperature is indicated above the panel, the duration of growing, at the bottom (hours). (WT) Wild-type strain W303; (+pE1 and +pE2) strains expressing Eno1p or Eno2p from centromeric plasmids pE1 or pE2. (B) The effect of ENO2 gene deletion or mutation on tRK1 import in vivo. Northern hybridization detection of tRK1 and control tRNAs [mitochondrial tRNA(Leu) and cytoplasmic nonimported tRNA(Lys), tRK2] are presented. On the left, M indicates RNA isolated from purified mitochondria, and T indicates total RNA. Strains and temperature of cultivation are indicated on top. (eno1/2) eno1 eno2 deletant strain; (pE2-H373F) plasmid expressing Eno2p with substitution H373F. Equal amounts of RNAs were analyzed for each strain. The ratio between the signals for tRK1 and mt tRNA (Leu) used as a criterion of import efficiency is presented in the middle. (C) Quantification of tRK1 import efficiency, respiration, and enolase enzymatic activity in the crude cell lysates of different strains (listed on the left side of the panel). (D) Effect of ENO2 gene deletion or mutation on mitochondrial translation. The autoradiograph of the PAGE-separated [³⁵S]-labeled mitochondrially synthesized polypeptides is presented. Strains and the temperature of incubation during the translation reaction are indicated above. Polypeptides were identified by comparison with standard mitochondrial translation pattern (McKee et al. 1984). (Cox 1, 2, and 3) Subunits 1–3 of cytochrome c oxidase; (Cyt b) cytochrome c; (ATPase 9) subunit 9 of ATP synthase. Additional bands appearing at 37°C with the H373F Eno2p version are indicated with the asterisks.