Cytosolic factor- and TOM-independent import of C-tail-anchored mitochondrial outer membrane proteins - PubMed (original) (raw)

Cytosolic factor- and TOM-independent import of C-tail-anchored mitochondrial outer membrane proteins

Kiyoko Setoguchi et al. EMBO J. 2006.

Abstract

C-tail-anchored (C-TA) proteins are anchored to specific organelle membranes by a single transmembrane segment (TMS) at the C-terminus, extruding the N-terminal functional domains into the cytoplasm in which the TMS and following basic segment function as the membrane-targeting signals. Here, we analyzed the import route of mitochondrial outer membrane (MOM) C-TA proteins, Bak, Bcl-XL, and Omp25, using digitonin-permeabilized HeLa cells, which provide specific and efficient import under competitive conditions. These experiments revealed that (i) C-TA proteins were imported to the MOM through a common pathway independent of the components of the preprotein translocase of the outer membrane, (ii) the C-TA protein-targeting signal functioned autonomously in the absence of cytoplasmic factors that specifically recognize the targeting signals and deliver the preproteins to the MOM, (iii) the function of a cytoplasmic chaperone was required if the cytoplasmic domains of the C-TA proteins assumed an import-incompetent conformation, and intriguingly, (iv) the MOM-targeting signal of Bak, in the context of the Bak molecule, required activation by the interaction of its cytoplasmic domain with VDAC2 before MOM targeting.

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Figures

Figure 1

Figure 1

Mitochondrial preprotein import in semi-intact cells. (A, B) HeLa cells grown on coverslips were semi-permeabilized with 25 μg/ml digitonin, and incubated at 26°C for 60 min with the reticulocyte lysate-synthesized 3FLAG-Bak (A) or Su9-DHFR-HA (B). After fixation and permeabilization with 1% Triton X-100 for 5 min, the cells were processed for indirect immunofluorescence microscopy as follows: all the cells were stained with monoclonal anti-FLAG (3FLAG-Bak) and anti-HA (Su9-DHFR-HA) antibodies, or anti-Tom22 (mitochondria), anti-Sec61β (ER), anti-giantin (Golgi), and anti-Pex14p (peroxisomes). 3FLAG-Bak and Su9-DHFR-HA are shown in red and organelle-specific marker proteins are shown in green. Merged images are also shown. × 4 magnification of the central area (outlined by a box) of the micrographs. Magnification, × 630; bar=20 μm. (C) The semi-intact cells were incubated with Su9-DHFR-HA and 3FLAG-Bak as described above. After fixation and 1% Triton X-100 permeabilization, the cells were incubated with rabbit anti-HA antibodies (green) and mouse monoclonal anti-FLAG antibodies (red). Boxed region in (C) is shown at threefold magnification. Note that rabbit polyclonal anti-HA antibodies stained nuclear matrix for unknown reasons. Bar=20 μm.

Figure 2

Figure 2

Competitive inhibition of mitochondrial import of C-TA proteins by an excess amount of 6myc-Bak. (A) Reticulocyte lysate-synthesized proteins used in this assay (3 μl each) were analyzed by SDS–PAGE and subsequent immunoblotting using the indicated antibodies. Substrate proteins are indicated by arrowheads. (B) The indicated precursor proteins (5 μl each) were subjected to mitochondrial import reaction (in 120 μl) at 26°C for 45 min in semi-intact cells in the presence or absence of an excess amount (40 μl) of 6myc-Bak. Other conditions were as in Figure 1. Imported preproteins and competitor 6myc-Bak are shown in green and red, respectively. (C) The extent of import was quantified by NIH Image, setting fluorescence intensities in the absence of 6myc-Bak at 100%. Three independent fields (each contains at least 100 cells) in a representative experiment were analyzed.

Figure 3

Figure 3

Mitochondrial import of C-TA proteins in the TOM component-depleted semi-intact cells. (A) Schematic representation of small interfering RNA (siRNA) transfection and import assay. (B) HeLa cells were subjected to siRNA transfection for the indicated proteins and following in vitro import assay using the semi-intact cells at 26°C for 60 min. The semi-intact cells were processed for double indirect immunofluorescence microscopy with either anti-HA antibodies (green; for Su9-DHFR-HA and HA-VDAC2) or anti-FLAG antibodies (green; for 3FLAG-Bak) and antibodies against the indicated Tom proteins (red). Merged images are also shown. (C) HeLa cells subjected to RNAi experiments as in (B) (equal protein amounts) were analyzed by SDS–PAGE and subsequent immunoblotting using the indicated antibodies.

Figure 4

Figure 4

Import of C-TA proteins into proteinase K-treated mitochondria in semi-intact cells. (A) Cartoon of the in vitro import assay using proteinase K-treated semi-intact cells. Red balls represent the cytoplasmic domains of membrane proteins. Green balls represent the imported C-TA proteins. (B) Semi-intact HeLa cells were treated with or without proteinase K as in (A) and processed for indirect immunofluorescence microscopy; the cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, and immunostained using antibodies against the indicated proteins. See Materials and methods for details. (C) Semi-intact cells were incubated with or without proteinase K under the indicated digitonin concentrations. All these samples were subjected to SDS–PAGE and subsequent immunoblot analysis using antibodies against the indicated proteins. (D) The indicated substrates were subjected to the import reaction at 26°C for 45 min in proteinase K-treated semi-intact cells. After the import reaction, the cells were processed for double indirect immunofluorescence microscopy with either anti-FLAG antibodies (green; 3FLAG-Bak, 3FLAG-Omp25, and 2FLAG-GST-Bcl-XL) or anti-HA antibodies (green; Su9-DHFR-HA) and antibodies against IMS protein HtrA2 (red). Merged images are also shown. (E) The cells shown in (D) were treated with 100 mM sodium carbonate (pH 11.5) to separate the supernatant (S) and membrane precipitates (P), which were analyzed by SDS–PAGE and subsequent immunoblotting using the indicated antibodies.

Figure 5

Figure 5

Mitochondrial import of Bak is compromised in VDAC2-depleted semi-intact cells. (A, B) The indicated preproteins were subjected to mitochondrial import in VDAC1- or VDAC2-depleted semi-intact (A) or intact (‘in vivo' (B)) cells. RNAi and the in vitro import assay were performed as described in Figure 3. Cells were processed for double indirect immunofluorescence microscopy with either anti-FLAG antibodies (green; 3FLAG-Bak, 3FLAG-Omp25, and 2FLAG-GST-Bcl-XL) or anti-Tom22 antibodies (red). Merged images are also shown. (C) Cell lysates (equal protein amounts) in (B) were analyzed by SDS–PAGE and subsequent immunoblotting (red) using antibodies against the indicated proteins. (D) The indicated preproteins were subjected to mitochondrial import in VDAC2-overexpressing semi-intact cells. The semi-intact cells were processed for double indirect immunofluorescence microscopy as in (A). The asterisks indicate cells overexpressing HA-VDAC2. (E) Cell lysates (equal protein amounts) prepared from (D) were analyzed by SDS–PAGE and subsequent immunoblotting using the antibodies against the indicated proteins.

Figure 6

Figure 6

Mitochondrial import of the PURESYSTEM-synthesized chimeric proteins consisting of GFP and MOM-targeting signals of C-TA proteins proceeds in the absence of cytoplasmic factors. (A) GFP-BakC, GFP-Bcl-XLC, and GFP-Omp25C were synthesized in the PURESYSTEM and subjected to mitochondrial import in semi-intact cells in the presence or absence of rabbit reticulocyte lysate (20 mg/ml (+RL)). Cells were processed for double indirect immunofluorescence microscopy with anti-GFP antibodies (green) and anti-Tom22 antibodies (red). Merged images are also shown. (B) HeLa cells were permeabilized with 25 μg/ml (lane 2) or 100 μg/ml digitonin (lane 3). After 5 min at 26°C, the semi-intact cells were washed and subjected to SDS–PAGE and subsequent immunoblot analysis using the indicated antibodies. (C) GFP-Pex26C was cotranslated with or without T7-tagged Pex19p in the PURESYSTEM. The reaction mixtures were analyzed by SDS–PAGE and subsequent immunoblotting using the indicated antibodies. (D) _In vitro_-synthesized GFP-Pex26C as in (C) was incubated with semi-intact HeLa cells and the cells were processed for double indirect immunofluorescence microscopy with anti-GFP antibodies (red) and anti-Pex14p antibodies (peroxisome marker; green). Merged images are also shown. (E) GFP-Bak was cotranslated in PURESYSTEM with (+) or without (−) Pex19p, then subjected to mitochondrial import as described in (D).

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