Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1

Abstract

In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock-down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.

Figure 1. The TbLOK1 protein is essential for trypanosome growth

A. Knockdown of TbLOK1 stops cell division. Cells were grown without (−RNAi) or with (+RNAi) induction of TbLOK1 RNAi, and then parasites were counted. Values on the Y-axis are the measured cell number times the dilution factor. B. TbLOK1 mRNA levels decrease following RNAi induction. At the indicated times, total RNA was electrophoresed, blotted and probed with TbLOK1 or the tubulin gene (load control). RNA from equivalent number of cells was loaded in each lane. C. The TbLOK1 protein levels drop following RNAi. At the indicated times, cells were isolated and extracted proteins were electrophoresed and western blotted with antibodies to TbLOK1, TbHSP70mt and enolase (a load control). Proteins from equivalent numbers of cells were loaded in each lane.

Figure 2. Knockdown of the TbLOK1 protein causes an incomplete loss of kDNA

A. TbLOK1-depleted cells lose kDNA. Uninduced cells (day 0) and cells induced with tetracycline to produce double-stranded RNA corresponding to TbLOK1 for 5 days were DAPI-stained and examined by phase contrast (left panels) and fluorescence (right panels) microscopy. K, kinetoplast. N, nucleus. B. Kinetics of kDNA loss. At indicated time of TbLOK1 RNAi, trypanosomes were fixed, stained with DAPI, and at least 100 cells at each time point were scored for either normal, small or absent kDNA. A typical fluorescence image of each category is shown above graph. White arrow points to a small kinetoplast. C. Minicircle and maxicircles decrease following LOK1 RNAi. Total trypanosome DNA was isolated at the indicated times following RNAi, digested with HindIII and XbaI, electrophoresed and Southern blotted. The nuclear-encoded trypanosome hexose transporter (THT) was the loading control.

Figure 3. TbLOK1 depletion leads to asymmetric kDNA division

A. Examples of cells undergoing symmetric (sym, left panels) or asymmetric (asym, right panels) kDNA division. Arrows indicate kDNA. B. Quantitation of cells undergoing symmetric (gray bars) or asymmetric (black bars) kDNA division during RNAi-mediated knockdown of TbLOK1. C. Quantitation of the number of cells with two kDNAs, in which the two kinetoplasts are connected by a maxicircle thread or nabelschnur (Gluenz et al., 2011, Gluenz et al., 2007) after RNAi of TbLOK1 as visualized by FISH with specific probes. D. Examples of FISH analysis with minicircle and maxicircle probes in cells containing maxicircle threads after knockdown of TbLOK1. Arrows indicate thread structures. K, kinetoplast; N, nucleus.

Figure 4. TbLOK1 is necessary for normal mitochondrial shape

A. TbLOK1 RNAi causes loss of mitochondrial structure. Uninduced cells (day 0), or those induced for TbLOK1 RNAi for 4 or 5 days were stained with MitoTracker Red (MiTr) and DAPI, decorated with antibodies to TbHSP70mt, and then examined by phase and fluorescence microscopy. B. Kinetics of mitochondrial changes following TbLOK1 depletion. Images from RNAi-treated cells (~100 cells from each time point) were examined as in A, and those with abnormal mitochondria (either unbranched organelles, or those that lost tubular mitochondria shape) were counted. The dotted line on the graph indicates the number of cells whose mitochondrion failed to stain with MitoTracker. C. Image of wild-type (strain 927) BSF cells stained with MitoTracker. D. Collapse of mitochondrial structure after prolonged TbLOK1 knockdown. After 5 days of TbLOK1 RNAi, trypanosomes were stained using antibodies to TbHSP70mt and imaged. The complete loss of a tubular-shaped mitochondrion in one cell is shown (white arrow).

Figure 5. Levels of TbLOK1 in different life cycle stages

A. Transcript abundance in wild-type 927 PCF and 427 BSF cells. RNA from equivalent number of cells was loaded in each lane, and tubulin was used as a loading control for Northern blots. B. Western blot comparing levels of TbLOK1, TbHSP70mt and enolase in PCF and BSF cells. Proteins from equivalent numbers of cells were loaded in each lane.

Figure 6. Ultrastructure of the mitochondrion is normal in TbLOK1-depleted cells

Uninduced cells (A, B) or those depleted of TbLOK1 for 4 days (C, D, F) were fixed, embedded in resin, and then thin sections were examined by EM. E. Enlargement of the area corresponding to the white box in D. K, kinetoplast; MM, mitochondrial membranes; black arrowheads, basal bodies; white arrowheads, cristae. Scale bar for A and F, 800 nm; for B, 300 nm; for C and E, 600 nm; for D, 3.0 μm

Figure 7. Disruption of kDNA replication or maxicircle transcription does not directly affect mitochondrial shape

Following RNAi to either DNA ligase kα (TbLIGkα) or mitochondrial topoisomerase II (TbTOP2mt), trypanosomes were stained with DAPI, MitoTracker (MiTr), and antibodies recognizing mtHSP70 and then examined by phase or fluorescence microscopy.

Figure 8. TbLOK1 knockdown inhibits mitochondrial respiration

TbLOK1 RNAi was induced for the indicated times and the rate of oxygen consumption measured. For comparison, cells knocked down for mitochondrial DNA ligase kα (TbLIGkα) were also evaluated.

Figure 9. TbLOK1 RNAi causes a defect in maxicircle transcription

A. After TbLOK1 depletion, mRNA was electrophoresed and northern blots probed for TbLOK1 (to show knockdown efficacy) and tubulin (a loading control), as well as the maxicircle-encoded 12S rRNA, cytochrome oxidase subunit I (CO1) and subunit II (CO2), NADH dehydrogenase subunit I (NDI) and subunit 7 (ND7), and the A6 subunit of the F₁/F_o-ATPase. B. Quantitation of the blots shown in A, normalized to nuclear-encoded tubulin. The amount of each transcript present at day 0 of RNAi was arbitrarily set to 100. C. Following RNAi-mediated knockdown of TbRNAPmt, trypanosomes were stained with MitoTracker (MiTr), subjected to immunofluorescence with antibodies recognizing mtHSP70, stained with DAPI, and examined by phase and fluorescence microscopy. Although TbRNAPmt RNAi induces maxicircle loss, minicircle levels are not initially affected (Grams et al., 2002). Thus, DAPI staining of the kinetoplast is not significantly reduced following TbRNAPmt depletion.

Figure 10. TbLOK1 is a mitochondrial membrane protein

A. Immunofluorescence studies show TbLOK1 is mitochondrial. Wild-type 927 cells were fixed and permeabilized, then decorated with antibodies to TbLOK1 and TbHSP70mt. Following DAPI staining, cells were examined by phase and fluorescence microscopy. B. Immunoelectron microscopy shows TbLOK1 at the mitochondrial membranes. Cryosections from trypanosomes where both alleles express the TbLOK1::myc fusion protein were fixed, and thin-sections decorated with antibodies to the myc epitope. Following incubation with gold-conjugated secondary antibodies, sections were osmium stained and examined by EM. Arrows indicate clusters of myc signal along the mitochondrial double membrane. C. TbLOK1 is an integral membrane protein. Mitochondria isolated from wild-type trypanosomes, or cells in which one allele expresses the TbLOK1::myc fusion, were treated with either buffer alone or with sodium carbonate, pH 11.5. Pellets (P) and supernatants (S) were analyzed by western blotting with antibodies to TbHSP70mt (a soluble protein), TAO (a membrane protein), or TbLOK1. Migration of TbLOK1::myc is indicated with an arrow.

Figure 11. TbLOK1 is a mitochondrial outer membrane protein

A. TbLOK1 is in the outer mitochondrial membrane. Wild-type cells were treated with either buffer, or with different concentrations of digitonin. Samples were centrifuged and the supernatants analyzed by western blotting with antibodies to TbLOK1 and the following marker proteins: cytochrome c (intermembrane space), TbHSP70mt (matrix), carnitine palmitoyl transferase (CPT, outer membrane), and trypanosome alternative oxidase (TAO, inner membrane). B. Darker exposure of the gel shown in ‘A’. C. TbLOK1 cofractionates with the outer membrane porin protein. Cells were treated with 0.04% digitonin, 0.1% digitonin, or with buffer alone, centrifuged and the supernatants blotted with antibodies to the TbLOK1, porin or TAO proteins. D. Protease digestions of isolated mitochondria. Mitochondria from 927 trypanosomes were treated with the indicated amounts of proteinase K and then Western blotted for TbLOK1, TbHSP70mt and cytochrome c. As indicated, some samples were treated with 1% Triton X-100 (TX-100) prior to the addition of protease.