OM14 is a mitochondrial receptor for cytosolic ribosomes that supports co-translational import into mitochondria - PubMed (original) (raw)

OM14 is a mitochondrial receptor for cytosolic ribosomes that supports co-translational import into mitochondria

Chen Lesnik et al. Nat Commun. 2014.

Erratum in

Abstract

It is well established that import of proteins into mitochondria can occur after their complete synthesis by cytosolic ribosomes. Recently, an additional model was revived, proposing that some proteins are imported co-translationally. This model entails association of ribosomes with the mitochondrial outer membrane, shown to be mediated through the ribosome-associated chaperone nascent chain-associated complex (NAC). However, the mitochondrial receptor of this complex is unknown. Here, we identify the Saccharomyces cerevisiae outer membrane protein OM14 as a receptor for NAC. OM14Δ mitochondria have significantly lower amounts of associated NAC and ribosomes, and ribosomes from NAC[Δ] cells have reduced levels of associated OM14. Importantly, mitochondrial import assays reveal a significant decrease in import efficiency into OM14Δ mitochondria, and OM14-dependent import necessitates NAC. Our results identify OM14 as the first mitochondrial receptor for ribosome-associated NAC and reveal its importance for import. These results provide a strong support for an additional, co-translational mode of import into mitochondria.

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Figures

Figure 1

Figure 1. OM14 interacts with NAC.

(a) Scheme of the PCA screen. Yeast strain expressing a bait protein fused to the amino (N)-terminal half of DHFR is mated with a library of yeast strains, each expressing a prey ORF fused to the carboxy (C)-terminal half of DHFR. Mating is done automatically, and the resulting cells are plated in an ordered manner of methotrexate-containing plates. Proximity of the two DHFR halves allows cells’ growth in the presence of methotrexate. Colonies size are determined with Balony program after 3 days of growth and compared with cells that express only the DHFR halves. (b) Example of selected interactions with NAC members, Egd1 and Egd2. Egd2 is known to heterodimerize with Egd1, Tom71 serves as a control for other mitochondria outer membrane proteins and Mck1 is a cytosolic kinase that serves as a standard negative control in such assays. (c,d) Co-immunoprecipitation analysis. Strains expressing either untagged or HA-tagged OM14 were subjected to immunoprecipitation (IP) with anti-HA beads, and samples from different steps of isolation were analysed by western blot with the indicated antibodies. Total indicates sample before mixing with the beads, Flow Thru. (flow through) is a sample from the unbound material, Wash indicates sample from the last wash of beads and IP is the eluted material from the beads. The histogram (d) presents the average results of NAC co-IP efficiency, calculated as the IP/input ratio for NAC divided by HA-OM14 ratio. Data are from at least three independent biological replicates (n), each entailing the entire procedure described above, from cell growth to western analysis. Error bars represent s.e.m. P value=0.028 (independent-samples one-sided _t_-test). Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov).

Figure 2

Figure 2. Decreased NAC and ribosomes association with OM14Δ mitochondria.

(a) Cells were subjected to cellular fractionation and samples from the cytosolic (Cyt.) or mitochondrial (Mito.) fractions were subjected to western analysis with the indicated antibodies. The histogram presents the average of the ratio between NAC signals in the two fractions from several independent biological repeats (n), each entailing the entire procedure, from cells’ growth to western analysis. Error bars represent s.e.m. P value was calculated by independent-samples one-sided _t_-test. Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov). (b) Purification of mitochondria depleted of ER or ribosomes. Following translational arrest, cell lysate (T) was prepared in the presence of EDTA and separated into cytosolic (C) and membrane (M) fractions by differential centrifugation. The M fraction was further separated through a sucrose gradient into six fractions. Aliquot from each fraction was subjected to western analysis with the indicated antibodies. (c) ER and ribosome-depleted mitochondria were isolated either from OM14+ or OM14Δ cells, incubated with ribosomes and subjected to centrifugation. Sup (S) and pellet (P) samples were analysed by western blot with the indicated antibodies. The arrow indicates the horizontal line in which the membrane was cut, to allow simultaneous incubation with the indicated primary antibodies. Also, unrelated lane was cropped between lanes 2 and 3. (d) Ribosomes with a nascent chain of mitochondria protein (MDH1) were incubated with mitochondria and separated to S and P, as above. Samples were analysed by western blot with the indicated antibodies. The presented panels are from the same image, that was cropped to remove unrelated lanes. Histograms present the results of three independent biological repeats and statistics were calculated as above. Y axis is the (P/P+S) signal for Rpl3, normalized to the signal of Tom20.

Figure 3

Figure 3. Lower OM14 association with NACΔ ribosomes.

Ribosomes were isolated from cells either expressing or deleted of NAC genes. A sample before isolation (total) or after centrifugation steps (ribosomes) was analysed by western analysis with the indicated antibodies. The histogram presents the average ratio of signals from several independent biological repeats (n) each entailing the entire procedure, from cells’ growth to western analysis. Error bars represent s.e.m. P value was calculated by independent-samples one-sided _t_-test. Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov).

Figure 4

Figure 4. OM14Δ mitochondria exhibit lower co-translational import efficiency.

(a–e) Co-translational import of MDH1t precursor: (a) Stalled ribosome–nascent chains complexes were isolated by centrifugation through a sucrose cushion and mixed with highly purified mitochondria. Ethidium bromide staining of pelleted RNCs is presented. Bands at the sizes of rRNAs of the small and large subunit are indicated by asterisks. (b) RNCs labelled by 35S-Met were mixed with highly purified mitochondria (fraction five in Fig. 2b) and incubated for 5 min with or without 20 mM EDTA. Mitochondria were then isolated by centrifugation and proteins associated with mitochondria (pellet) or not (sup) were resolved on PAGE. The arrow indicates a protein that was inserted into the mitochondria and cleaved (hence it is shorter). This band is not detected in the (−Δ_Ψ_) control reaction in which the membrane potential was diminished. Input panel is a sample from the protein labelling reaction before mixing with mitochondria. (c) MDH1t RNCs were mixed with highly purified mitochondria from OM14 + or OM14Δ cells. At the indicated time points, an aliquot was set aside and resolved on PAGE and phosphorimager. Control reactions included the addition of 1 μg ml−1 of valinomycin (−Δ_Ψ_) during a 10 min reaction or addition of proteinase K (pK) at the end of a 10 min import reaction, to remove non-imported bands. (d) Import assays were repeated three times (n), with three time-point measurements in each. Every repeat entailed a new mitochondria prep and a new RNC prep. The average value and s.e.m. for each time point is presented. Graphs are the best-fit linear slope. (e) The histogram presents the average and s.e.m of the best-fit slopes from the three (n) independent experiments. P value was calculated by independent-samples one-sided _t_-test. Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov). (f,g) Post-translational import: Full-length MDH1 (MDH1f) was synthesized from its normal ORF in a rabbit reticulocyte lysates with 35S-Met. Import assays were performed as above, with the indicated controls. Panel e presented the results of three biological repeats, with three time-point measurements in each. Every repeat entailed new mitochondria prep and a new protein synthesis reaction. The average value and s.e.m. for each time point is presented. Graphs are the best-fit linear slope. No statistically significant difference is apparent between the linear best fits of graphs (P value=0.33, independent-samples one-sided _t_-test).

Figure 5

Figure 5. OM14 necessitates NAC to exert its role.

(a–c) NAC from rabbit reticulocyte lysates (RRL): MDH1t RNCs depleted of NAC were mixed with mitochondria purified from either OM14 + or OM14Δ cells. The import reactions were supplemented with NAC-containing high-salt supernatant (HS) from RRL, and at the indicated time points a sample was collected. The arrow indicates the imported protein. Import assays were repeated four times, each time with a new mitochondria prep and a new RNC prep. Graphs (b), histogram (c) and statistics were calculated as described for Fig. 4. (d–f) NAC from yeast: Import assays entailing NAC-depleted RNCs and OM14+ or OM14Δ mitochondria were supplemented with ribosome-associated factors, prepared from NAC+ yeast strain. Graphs (e), histogram (f) and statistics were calculated as described for Fig. 4. (g) NAC purified from bacteria: Import assays entailing OM14+ or _OM14_Δ mitochondria and MDH1t RNCs depleted of NAC, were supplemented with NAC complex that was expressed and purified to a high degree from bacteria (pur. NAC). Samples were collected at the indicated time points, resolved on PAGE and exposed to phosphorimager for 3 days. Panel is from a single image (provided as Supplementary Fig. 4e), which was cropped between lanes 4 and 5 to remove irrelevant lanes.

Figure 6

Figure 6. Two modes for protein import into mitochondria.

Depicted to the left is the well-established post-translational import mode, in which chaperones (circles) assist fully translated proteins (black line) in their transport through the TOM complex into the mitochondria. Our results corroborate an additional mode, described on the right. In this model, OM14 interacts with the heterodimeric NAC, while it is associated with translating ribosomes. This interaction brings the emerging protein to proximity with the TOM complex thereby enhancing import. These modes are not mutually exclusive and likely to be redundant under many experimental conditions.

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