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. 2012 Aug 10;287(33):27467-79.
doi: 10.1074/jbc.M112.382093. Epub 2012 Jun 28.

Chloroplast β-barrel proteins are assembled into the mitochondrial outer membrane in a process that depends on the TOM and TOB complexes

Thomas Ulrich  1 , Lucia E Gross, Maik S Sommer, Enrico Schleiff, Doron Rapaport
Affiliations

Chloroplast β-barrel proteins are assembled into the mitochondrial outer membrane in a process that depends on the TOM and TOB complexes

Thomas Ulrich et al. J Biol Chem. .

Abstract

Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.

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Figures

FIGURE 1.
FIGURE 1.
Chloroplast β-barrel protein Oep37 is exclusively targeted in vivo to chloroplast outer envelopes and possesses an Nin-Cin topology. The indicated constructs were co-transfected into A. thaliana protoplasts that were subsequently analyzed by confocal fluorescence microscopy. The GFP fluorescence (GFP), the autofluorescence of chlorophyll, the overlay of all fluorescence signals, and the differential interference contrast image (DIC) are shown for a representative example. A, two saGFP fragments were targeted to the cytoplasm (CYT-S11 and CYT(1–10), top panel), to the chloroplasts IMS (Tic22-S11 and Mgd1(S1–10), middle panel), or to the outer envelope of chloroplasts (Oep7-S11 and CYT(S1–10), bottom panel). B, two saGFP fragments were targeted to the mitochondrial IMS (S11-VDAC3 and Tim50(S1–10)). A staining of mitochondria with MitoTracker is shown. C, Oep37 either N- or C-terminally fused to saGFP11 (S11-Oep37 or Oep37-S11, respectively) was co-expressed with the chloroplast IMS-located Mgd1(S1–10). The middle panel shows an isolated chloroplast after osmolysis of protoplasts. D, S11-Oep37 fusion proteins were co-expressed with either cytosolically localized S1–10 (CYT(1–10)) or with S1–10 located in the mitochondrial IMS (Tim50(S1–10)). As another control, VDAC3 fused to saGFP11 (S11-VDAC3) was co-expressed with the chloroplast IMS-located Mgd1(S1–10).
FIGURE 2.
FIGURE 2.
GFP signal is not observed when the two fragments are not in the same compartment. The indicated constructs were co-transfected into A. thaliana protoplasts that were subsequently analyzed by confocal fluorescence microscopy. The GFP fluorescence after GFP assembly (GFP), the autofluorescence of chlorophyll, and the differential interference contrast image (DIC) are shown for a representative example. A, large fragment was targeted to the chloroplast IMS (MGD1(S1–10)), whereas the small one was targeted to either the cytosol (CYT-S11) or the chloroplast stroma (pSSU-S11). B, large fragment was targeted to the mitochondrial IMS (Tim50(S1–10)), whereas the small one was targeted to either the cytosol (CYT-S11) or the mitochondrial matrix (F1β-S11).
FIGURE 3.
FIGURE 3.
Oep37 can be imported in vitro into chloroplasts and mitochondria. Radiolabeled Oep37 was incubated for 30 min at RT with either isolated pea chloroplasts (A) or isolated mitochondria (B). Membrane insertion was assayed by the resistance to carbonate treatment (CO32−) in the presence or absence of Triton X-100 (Tx). An aliquot (5%) of the translation product (TP) used for each import reaction was loaded as control. The bands corresponding to Oep37 are indicated with an arrowhead. C, purity of the isolated mitochondria (M) or chloroplasts (C) was monitored by analyzing 10 μg of total organellar protein by SDS-PAGE followed by immunodecoration, using organelle-specific antibodies (α-VDAC for mitochondria and α-Toc75 for chloroplasts). The bands corresponding to the marker proteins are indicated with an arrowhead, whereas unspecific bands resulting from cross-reactivity of the antibodies are marked with an asterisk.
FIGURE 4.
FIGURE 4.
Oep37 and Oep24 are targeted in vitro in a dual system to the OM of mitochondria and chloroplasts. A, single (lanes 2–4) and dual (lanes 5–13) import of rabbit reticulocyte-translated pOE33 or pALDH into isolated pea chloroplasts (chloro) (lanes 2–6, 10, and 11) and mitochondria (mito) (lanes 7 and 8 and 12 and 13). Translation products (TP, 10%) as input control are shown in lanes 1 and 9. Nonimported proteins were removed by thermolysin (TH, lanes 3, 6, 8, 11, and 13). In addition, 1% (v/v) Triton X-100 was added to one sample (TX, lane 4). Precursor and mature forms of pOE33 and pALDH are indicated by white and black arrowheads, respectively. The stromal intermediate form of pOE33 is indicated with a gray arrowhead. B, purity of the chloroplasts (lanes 1 and 2) and mitochondrial (lanes 3 and 4) fractions after dual import and re-purification of the organelles was assayed by Western blotting with the indicated organelle-specific antibodies. C, chloroplast outer envelope membranes were treated with the indicated concentrations of either thermolysin (lanes 1–4) or proteinase K (lanes 5–8). The membranes were then assayed by Western blotting using the indicated organelle-specific antibodies. Oep37 was partially sensitive to high amounts of proteinase K, and in addition to full-length protein, a slightly smaller degradation product was also observed (lane 5, white and black arrowheads, respectively). D–F, dual import of radiolabeled Oep37 (D), Oep24 (E), and VDAC1 (F) into chloroplasts and mitochondria (lanes 2–11). Translation products (TP, 10%) as input control are shown in lane 1. Nonimported proteins were removed by either thermolysin (TH, lanes 3 and 8) or proteinase K (PK, lanes 4 and 9) treatment. Full integration into the membrane was assayed by carbonate extraction (EX) in the presence or absence of Triton X-100 (TX, lanes 5, 6, 10, and 11).
FIGURE 5.
FIGURE 5.
Oep37 expressed in yeast cells is assembled into the mitochondrial OM in a native conformation. A, Oep37 is located in mitochondria. Lysates of Oep37-expressing cells and fractions corresponding to mitochondria, endoplasmic reticulum (ER), and cytosol were analyzed by SDS-PAGE and immunodecoration with antibodies against Oep37, the mitochondrial protein Tom70, a marker protein for the cytosol (hexokinase), and the endoplasmic reticulum protein Erv2. Mitochondria isolated from untransformed WT cells were co-analyzed as a control. B, mitochondria isolated from cells expressing Oep37 were analyzed directly by SDS-PAGE (input) or were subjected first to carbonate extraction and then centrifuged to discriminate between membrane proteins in the pellet and soluble proteins in the supernatant (SN). Additional aliquots of mitochondria were left intact or were treated with the indicated amounts of proteinase K. Proteins were analyzed by SDS-PAGE and immunodecorated with antibodies against the indicated proteins as follows: Tom70, an OM protein exposed to the cytosol; Porin, a protein embedded in the OM; Hep1, a mitochondrial soluble matrix protein; Mcr1, a protein with two isoforms, a 34-kDa species exposed on the OM and a 32-kDa soluble one in the IMS. C, mitochondria isolated from cells expressing Oep37 were left intact (total) or were treated with the indicated amounts of proteinase K (left panel) or trypsin (right panel). In one sample, the mitochondria were swelled (+SW) before the treatment with PK. Proteins were analyzed by SDS-PAGE and immunodecorated with antibodies against the indicated proteins. Tob55, a protein embedded in the OM; Dld1, an inner membrane protein exposed to the IMS. Proteolytic fragments are indicated with an asterisk.
FIGURE 6.
FIGURE 6.
Overexpression of Oep37 can partially complement the por1Δ phenotype. A, expression of Oep37 does not interfere with growth on a nonfermentable carbon source. Cells harboring either a plasmid encoding Oep37 or an empty plasmid (Ø) as control were tested by drop dilution assay for their ability to grow on glycerol-containing medium (YPG) at 30 and 37 °C. B, wild-type cells harboring an empty plasmid (Ø) and cells deleted for POR1 (por1Δ) that were transformed with either an empty plasmid (Ø) or a plasmid encoding Oep37 were tested by drop dilution assay for their ability to grow at 30 °C on rich (YPG) or synthetic glycerol-containing medium (SG-Leu).
FIGURE 7.
FIGURE 7.
Oep37 and Oep24 require the mitochondrial import receptors for their assembly into the OM. A, isolated mitochondria were left intact or pretreated with trypsin followed by re-isolation of the organelles. Next, radiolabeled precursor of either Oep37 or Oep24 was incubated with the trypsin-treated or intact mitochondria for the indicated time periods. At the end of the import reactions, samples were treated with PK, and proteins were analyzed by SDS-PAGE and autoradiography. The insertion of the proteins was quantified by analyzing the PK-protected molecules. The amount of precursor proteins imported into intact mitochondria for 20 min was set to 100%. An autoradiographic representative of three independent repeats and quantification of three independent experiments is presented. B, radiolabeled precursors of Oep37, Oep24, and Porin (as a control) were imported into mitochondria isolated from either tom20Δ or tom70/71Δ and their corresponding WT strains. Imported proteins were analyzed and quantified as described in the legend to A. C and D, mitochondria isolated from nontransformed WT cells and those isolated from either tom20Δ or tom70/71Δ and their corresponding WT strains transformed with either Oep37 (C) or Oep24 (D) encoding plasmid were analyzed by SDS-PAGE and immunodecoration with antibodies against Oep37 or Oep24, respectively. In addition, immunodecoration with antibodies against the indicated mitochondrial proteins was performed.
FIGURE 8.
FIGURE 8.
Integration of Oep37 and Oep24 into the mitochondrial OM requires the TOM import pore. Radiolabeled precursors of Oep37, Oep24, and Porin (as a control) were imported into mitochondria in the absence or presence of the indicated amounts of recombinant pSu9-DHFR. Imported proteins were analyzed and quantified as described in the legend to Fig. 7A. The amount of precursor proteins imported into mitochondria without added pSu9-DHFR was set to 100%.
FIGURE 9.
FIGURE 9.
Small Tim chaperones have only a minor role in the assembly of Oep37 and Oep24 into the mitochondrial OM. A, rupturing of the outer membrane does not compromise the assembly of Oep37. Radiolabeled precursors of Oep37 and AAC (as a control) were incubated for the indicated time periods with isolated intact mitochondria or with mitochondria that had been subjected to osmotic swelling. After import, mitochondria were pelleted and subjected to alkaline extraction, and the pellet fractions were analyzed by SDS-PAGE followed by autoradiography. The amount of precursor proteins imported into intact mitochondria for 20 min was set to 100%. An autoradiographic representative of three independent repeats and quantification of three independent experiments are presented. B, insertion of Oep37 and Oep24 is hardly affected in mitochondria lacking the Tim8/Tim13 complex. Radiolabeled precursors of Oep37, Oep24, and Porin were imported into mitochondria isolated from either tim8Δ/tim13Δ or its corresponding parental strain. Imported proteins were analyzed and quantified as described in the legends to Fig. 7A. C, insertion of Oep37 is not affected in mitochondria mutated in TIM10. Radiolabeled precursors of Oep37 and AAC were imported into mitochondria isolated from a strain harboring a temperature-sensitive allele of TIM10 (TIM10-1 (58)) or from its corresponding parental strain. Imported proteins were analyzed and quantified as described in the legends to Fig. 7A. D, mitochondria isolated from nontransformed WT cells and those isolated from either tim8/tim13Δ or TIM10-1 and their corresponding parental strains transformed with Oep37- or Oep24-encoding plasmid were analyzed by SDS-PAGE and immunodecoration with antibodies against Oep37 or Oep24, respectively. In addition, immunodecoration with antibodies against the indicated mitochondrial proteins was performed.
FIGURE 10.
FIGURE 10.
TOB complex is crucial for the mitochondrial integration of Oep37 and Oep24. A, radiolabeled precursors of Oep37, Oep24, and Porin were imported into mitochondria isolated from either WT or mas37Δ strains. Imported proteins were analyzed and quantified as described in the legends to Fig. 7A. B, mitochondria isolated from nontransformed WT cells and those isolated from mas37Δ and their corresponding wild-type cells transformed with either Oep37- or Oep24-encoding plasmid were analyzed by SDS-PAGE and immunodecoration with antibodies against Oep37 or Oep24, respectively. In addition, immunodecoration with antibodies against the indicated mitochondrial proteins was performed. C, Oep37 was transformed into cells expressing Tob55 under the control of the GAL10 promoter. Cells were harvested at the indicated time points after a shift from galactose- to glucose-containing medium. Crude mitochondria were isolated, and proteins were analyzed by SDS-PAGE and immunodecoration with antibodies against Oep37 and the indicated mitochondrial proteins. Tob55, Tom40, and Porin are β-barrel proteins.
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