Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR

Abstract

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.

Keywords: Trypanosoma brucei; evolution; membrane biogenesis; mitochondrial transport; outer membrane.

FIGURE 1.

RNAi-mediated ablation of POMP10 did not affect growth or mitochondrial morphology. A, growth curve of uninduced tetracycline (−Tet) and induced (+Tet) POMP10-RNAi cell line. Inset, Northern blot of the POMP10 mRNA after 2 days of RNAi induction. Ethidium bromide-stained gel showing the rRNAs served as a loading control. B, immunofluorescence analysis of induced (2 d and 7 d) and uninduced RNAi cells. Cells were stained with ATOM40 antiserum as a mitochondrial marker (red) and 4′,6-diamidin-2-phenylindol (DAPI) to highlight nuclear and mitochondrial DNA (blue). DIC, differential interference contrast.

FIGURE 2.

POMP10 is a signal-anchored mitochondrial OM protein. A, upper panel, outline of the mitochondrial purification scheme. Lower panel, immunoblot analysis of the indicated fractions of a mitochondrial purification from the cell line expressing full-length POMP10 that is C-terminally tagged with GFP (POMP10-GFP). The fractions (5 μg of each) depicted are whole cells (wc), cytosol (cyto), crude mitochondria (crude), and pure mitochondria (pure). The samples were separated by SDS-PAGE, blotted, and probed for GFP, the mitochondrial markers ATOM40, cytochrome c (Cyt c), and LipDH and the cytosolic marker EF1α. B, immunoblots of a protease protection assay using gradient-purified structurally intact mitochondria isolated from cell lines expressing POMP10-GFP analyzed by anti-GFP antiserum. The additions of proteinase K and of Triton X-100 are indicated. The OM protein ATOM69 served as a control OM protein. The IMS protein Tim9 and the matrix protein mtHsp70 served as controls for OM integrity. In the presence of Triton X-100 mtHsp70 was degraded to a protease-resistant fragment.

FIGURE 3.

Transgenic cell lines expressing POMP10-GFP fusions. A, FL, full-length GFP fusion. For the other fusion proteins the extent of the deletions (Δ) as well as the positions that were replaced with either serine (S) or glutamate (E) are indicated on the left. Amino acid sequences of the signal-anchor domains in the indicated POMP10-GFP fusion proteins are shown in the middle. The predicted membrane-spanning domain is underlined. The serine and glutamate residues that were used to replace the positively charged flanking amino acids are indicated in bold. The symbols used for the different GFP constructs are shown on the right: black line, cytosolic domain; box, signal-anchor domain; large white box, predicted membrane-spanning domain; small black boxes, positively charged flanking sequence(s); small white boxes, uncharged flanking sequence(s); small gray boxes, negatively charged flanking sequences. B, immunoblot analysis of total cellular extracts from equal cell numbers of the parental strain T. brucei 29-13 and the indicated transgenic cell lines expressing the various POMP10-GFP fusion proteins were probed for GFP and EF1α, which serves as a loading control.

FIGURE 4.

Characterization of the targeting signal in POMP10. IF analysis of the indicated POMP10-GFP fusion proteins (A–H, left columns) in procyclic forms of transgenic T. brucei (A–H, middle column). Samples were probed for GFP and the mitochondrial marker ATOM40, respectively. An overlap of both signals is also shown (Merge). Bar, 10 μm. Crude digitonin-based cell fractionation analysis (A–H, right column) of cell lines expressing the indicated POMP10-GFP fusion proteins. Total cellular extract (T), crude mitochondrial fraction (P), and digitonin-extracted cytosolic fraction (S) and shown. LipDH and EF1α served as mitochondrial and cytosolic markers, respectively. FL, full-length.

FIGURE 5.

Carbonate extractions at high pH of membrane-associated POMP10-GFP fusion proteins. Immunoblots of equal cells equivalents of total (T), pellet (P), and supernatant (S) fractions of a carbonate-extracted crude mitochondrial pellet obtained by digitonin extraction of cells expressing the indicated POMP10-GFP variants (A–E). The extraction was performed at both pH 10.8 and pH 11.5, and immunoblots were analyzed by anti-GFP. ATOM40 and Cyt c served as markers representing an integral and peripheral membrane protein, respectively.

FIGURE 6.

Expression of some of the truncated POMP10-GFP fusions interferes with growth. Growth curves of uninduced (black line) and induced (red line) cell lines expressing the indicated recombinant POMP10-GFP fusion proteins. All analyzed cell lines show an altered mitochondrial morphology (see Fig. 4, D–F). Insets, immunoblots confirming expression of the GFP fusion proteins. VDAC serves as loading control. Tet, tetracycline.

FIGURE 7.

In vivo biogenesis of POMP10 depends on pATOM36. A, IF analysis of full-length POMP10 that is C-terminally tagged with GFP (GFP(FL): green) and mitochondrial marker proteins (VDAC and ATOM69: red) in tetracycline (tet)-inducible pATOM36 (left panels), ATOM40 (middle panels), and SAM50 (right panels) RNAi cell lines. Time of induction in days (d) is indicated at the top. Bar, 10 μm. B, growth curves of tetracycline-inducible pATOM36 (left panels), ATOM40 (middle panels), and SAM50 (right panels) RNAi cell lines that co-express full-length POMP10-GFP and that were used in A. C, equal cell equivalents of the same RNAi cell lines shown in A (left panel, pATOM36; middle panel, ATOM40; right panel, SAM50) were analyzed for the presence or absence of pATOM36, ATOM40, ATOM14, ATOM46, ATOM69, VDAC, and cytosolic EF1a using immunoblots. Time of RNAi induction is indicated at the top of each panel. D, crude digitonin-based cell fractionation analysis of the same cell lines shown in A and B. Total cellular extract (top panels), crude mitochondrial fraction (pellet, middle panels), and digitonin-extracted cytosolic fraction (SN, bottom panels) were probed for POMP10-GFP (GFP(FL)). LipDH and EF1α served as the mitochondrial and cytosolic markers, respectively. E, same as D, but a tetracycline-inducible pATOM36 RNAi cell line that co-expresses the POMP10 fusion protein in which the transmembrane domain including the positively charged flanking residues was directly fused to the GFP was analyzed.

FIGURE 8.

Ablation of pATOM36 did not cause mislocalization to the ER. IF analysis of full-length POMP10 that is C-terminally tagged with GFP (POMP(FL): green) and the ER marker protein BiP (red) in the tetracycline-inducible pATOM36 RNAi cell line. Time of induction in days (d) is indicated at the top. Bar, 10 μm.

FIGURE 9.

In vitro biogenesis of POMP10 depends on pATOM36. A, left panel, in vitro insertion assay of the [³⁵S]Met-labeled POMP10-GFP(Δ33–560) using mitochondria isolated from the uninduced tetracycline (−Tet) and induced (+Tet, 2 days) procyclic pATOM36-RNAi cell line. Incubation times are indicated at the top. The pellet fraction of an alkaline carbonate extraction was separated by SDS-PAGE and analyzed by autoradiography. Lower panel, the section of the Coomassie-stained gel served as loading controls. Graph on the right, quantification of triplicate experiments shown on the left. Standard errors are indicated. B, graph of triplicate in vitro insertion assays (9-min time point) using isolated mitochondria of uninduced and induced pATOM36 and ATOM40 RNAi cell lines, respectively. The tested substrates are indicated at the top. Standard errors are indicated. The SDS-gels representing typical experiments including their corresponding loading controls are indicated below the graphs. Bottom panels, immunoblot confirming the specific down-regulation of pATOM36 and ATOM40 in the respective RNAi cell lines.