The S. cerevisiae strains used in the present study were isogenic to the wild type Σ1278b strain: 23344c (ura3, lab collection: Laboratory of Membrane Transport Biology, IBMM, Université Libre de Bruxelles) and 27061b (ura3 and trp1, lab collection: Laboratory of Membrane Transport Biology, IBMM, Université Libre de Bruxelles) (9,10). Cells were grown in a minimum buffered (pH 6.1) medium prepared as described previously (11) with 3% galactose (MGal), glucose (MGlu) or glycerol (MGly) as the carbon source or in non-inducible medium raffinose (MRaf) and glutamate as a nitrogen source, supplemented with vitamins and minerals (11). Yeasts were grown at 29°C.

For APOL1 expression, the human APOL1 was cloned into the centromeric p416Gal.1 (HA) (12) yeast expression plasmid under the control of a GAL1 promoter by homologous recombination. Yeast cells transformed with empty p416Gal.1 (HA) vector were used as control. The primers used are listed in Table I. The APOL with the mutated BH3 domain was amplified using PCR with Phusion^™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Inc.) and the APOL1-∆BH3F/R oligonucleotides (Table I) and introduced in the APOL1-pGal.1 vector. The reaction mixtures were processed with an initial denaturation period at 98°C for 30 sec, followed by a three-step PCR program for 30 cycles that consisted of 98°C for 10 sec, 70°C for 20 sec and 72°C for 25 sec, prior to a final extension step at 72°C for 10 min.

The mutant APOL1-∆BH3 (BH3 domain deletion) was sequenced by Beckman Coulter (Illumina HiSeq) to verify the introduction of the desired substitution. pYX232-mtGFP (TRP1), encoding green fluorescent protein (GFP) fused to the mitochondrial pre-sequence subunit 9 of the F₀-ATPase (mt-GFP) under the control of the constitutive triosephosphate isomerase promoter was used to monitor mitochondrial structure and morphology. pYX232-mtGFP was kindly provided by Professor Benedikt Westermann (Universität Bayreuth, Germany) (13).

Total protein extracts were performed as previously described (14). Aliquots of 1 ml at an optical density (OD) of 0.2 (10⁷ cells/ml; λ=660 nm) from cells at the exponential growth phase were harvested by centrifugation at room temperature for 5 min at 3,500 × g. The cell pellet was suspended in 500 µl water and the cells were lysed with 50 µl of 2 M NaOH for 10 min. The proteins were precipitated with 50 µl of 50% trichloroacetic acid and collected by centrifugation at 4°C for 5 min at 12,000 × g. The pellet was suspended with gel loading buffer containing 4% (w/v) SDS, 100 mM Tris pH 6.8, β-mercaptoethanol 2% (v/v), 20% (v/v) glycerol and blue bromophenol. Samples were heat-treated at 37°C for 10 min. For the western blotting analysis, equal protein amounts (~20 µg) were loaded onto a 8% SDS-PAGE. After transfer to nitrocellulose membranes, HA-tagged APOL1 was probed with anti-HA (1:10,000, cat. no. 26183, Molecular Probes; Thermo Fisher Scientific, Inc.), while Sc-Dpm1, Sc-Pma and Sc-porin were probed with anti-Dpm1 (1:5,000, cat. no. A-6429), anti-Pma1 (1:10,000, cat. no. MA1-91567) and anti-porin (1:5,000, cat. no. 459500; all from Invitrogen; Thermo Fisher Scientific, Inc.). Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies anti-rabbit-IgG (1:1,000, cat. no. NA934; Cytiva) and anti-mouse-IgG (1:5,000, cat. no. NA931; Cytiva) for 1 h at room temperature) followed by measurement of chemo-luminescence (Lumi-Light^PLUS, Roche Diagnostics). The western blot experiments in the present study analyzed membrane proteins; accordingly, Pma1 was used as a suitable control (15).

For growth in liquid medium, yeast were grown overnight at 29°C in MGlu or MGal medium and diluted to an OD (λ=660 nm) of 0.2 in MGlu and MGal, respectively. OD was measured repeatedly over 9 days. In order to test the proliferation of cells grown on agar plates, yeast previously grown in liquid MGlu or MGal were spotted at a final concentration of 10³ cells on MGlu or MGal solid media. In parallel, aliquots were collected for immunostaining for the detection of APOL1/APOL1-∆BH3 expression in inducible conditions.

Cells were cultured in MGal overnight at 29°C and then diluted in fresh media to an OD (λ=660 nm) of 0.2. Cell aliquots were collected, washed twice in PBS and re-suspended at 1×10⁷ cells/ml in 1 ml of 2.5 µg/ml dihydroethidium (DHE) (37291, Sigma-Aldrich) in PBS and incubated for 15 min in the dark at room temperature. Then, cells were washed with 1 ml PBS and analyzed via flow cytometry. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and results were analyzed with the FlowJo program (V10.5.0, FlowJo LLC).

Yeast strain cell cultures were collected at OD=0.2 (λ=660 nm) of the exponential phase in MGal medium. Cells were incubated with 50 pM TMRE (TMRE-Mitochondrial Membrane Assay Kit, ab113852, Abcam) for 10 min at 4°C, washed twice with PBS 1X, then cells were harvested, centrifuged (1,600 × g for 4 min at 4°C) and suspended (~1×10⁶ cells ml⁻¹) in PBS, in order to be analyzed by flow cytometry, according to the manufacturer's protocol, on a Canto II (BD Biosciences). Carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP, ab120081, Abcam) treatment, an uncoupling agent, was used as a positive control, as it is able to completely depolarize the mitochondrial outer membrane. It was added to cell cultures at a final concentration of 20 µM 20 min prior to the incubation with TMRE.

For vacuole staining, cells were collected at OD=0.2 (λ=660 nm) of the exponential phase in MGAl growth medium and resuspended in 1 ml of pre-heated medium and 80 nM FM4-64 (T3166, Invitrogen, Thermo Fisher Scientific, Inc.). Cells were incubated at 29°C for 15 min, centrifuged (5,000 × g at room temperature for 5 min), collected and resuspended in 5 ml of medium and incubated at 29°C for 120 min. For fixed (1.1 ml of 37% formaldehyde incubated at 29°C for 30 min) or live cells, mt-GFP was monitored at a wavelength of 510 nm (magnification, ×63 and ×100).

Yeast strains were cultured at 29°C overnight in MGal media until they reached the log growth phase. At OD=0.2 (λ=660 nm), cell aliquots of 9 ml were fixed with 1.1 ml of formaldehyde 37% and incubated at 29°C for 30 min. Cells were collected, washed and resuspended in Buffer B [Sorbitol (1 M), K₂HPO₄, 3H₂O (1 M) and KH₂PO₄ (1 M) at pH 7.5]. A total of 5 µl of lyticase (10,000 u/ml) and 2 µl of β-mercaptoethanol were added. After 30 min incubation at 29°C, cells were collected, washed and suspended in 1 ml of PBS 1X. A total of 20 µl of yeast culture was seeded on 10-well slides (MP Biomedicals, LLC.). Cells were stained for APOL1/APOL1-∆BH3 expression levels with mouse anti-HA (1:100, cat. no. 26183, Molecular Probes; Thermo Fisher Scientific, Inc.) overnight at room temperature in a humidified chamber. A series of PBS washes preceded the secondary anti-mouse antibody (1:1,500, A-11001; Invitrogen; Thermo Fisher Scientific, Inc.) incubation for 1 h at room temperature in a humid chamber. DAPI (cat. no. D9542; Sigma Aldrich) was used to stain the nuclei of yeast cells. The revelation of APOL1/APOL1-∆BH3 expression was assessed using a Zeiss inverted fluorescence microscope (magnification, ×63 and ×100).

The procedure was performed as previously described (16). Yeast strains were grown at 29°C for 16 h, collected by centrifugation (3,000 × g for 5 min at room temperature) and then washed twice with distilled water. Cells were suspended in dithiothreitol (DTT) buffer (100 mM Tris, pH 9.4, 10 mM DTT) and incubated at 29°C for 20 min with gentle shaking. After removing the supernatant by centrifugation at 3,000 × g for 5 min at room temperature, cells were resuspended in Zymolyase buffer (1.2 M sorbitol buffer containing 1 mg zymolyase 20T per gram of cells). Following incubation at 29°C for 30 min with gentle shaking, cells were centrifuged at 2,200 × g for 8 min and resuspended in homogenization buffer [0.6 M sorbitol, 1 mM EDTA, 10 mM Tris, 0.2% (w/v) BSA pH 6.0]. These cells were centrifuged at 2,200 × g for 8 min at 4°C and then homogenized 15 times with a tight pestle in a glass homogenizer. The unbroken cells were then pelleted at 1,500 × g for 5 min at 4°C. The resulting supernatant was collected and centrifuged first at 3,000 × g for 5 min at 4°C and again at 12,000 × g for 15 min at 4°C. After repeating centrifugation (3,000 × g for 5 min and 12,000 × g for 15 min at 4°C) and resuspension of this pellet to yield pure mitochondria, the mitochondrial pellet was finally resuspended in SEM buffer (10 mM MOPS, 250 mM sucrose, 1 mM EDTA pH 7.4). Highly pure enriched mitochondria fraction was subjected to a sucrose gradient purification. After centrifugation of 134,000 × g for 1 h at 4°C, an oxidized band was extracted and pelleted again at 10,000 × g for 30 min at 4°C. Western blot analyses were performed to examine preparation of the mitochondrial fraction as detailed above.

Exponentially MGal-growing cells at a final density of 10³ cells were spread onto MGly agar plates or MGal in parallel, incubated at 29°C for three days. At the end of the incubation, cells were observed and counted manually, and. the IRC was calculated as colony number observed on MGly plates divided by the number of colonies on MGal plates.

PRISM (version 6.0 GraphPad Prism software, Inc.) was used to perform statistical data analysis and plot the graphs. Data are presented as the mean ± standard error of the mean (SEM). A minimum of three experiments were performed for statistical analysis. One-way ANOVA followed by Tukey's post hoc test and Student's t-test were used to determine statistically significant differences between the different experimental conditions. P<0.001 was considered to indicate a statistically significant difference.

In order to investigate the action of human APOL1 in yeast, a sequence covering the whole APOL1 open reading frame was inserted in the yeast vector (pGAL1) allowing expression induction by galactose and repression by glucose. As the BH3 domain is one of the primary features of APOL1 and has been associated with the pro-apoptotic function of Bcl2 family proteins, the activity of the BH3-deleted version of APOL1 (∆BH3) was tested. Cells were cultured in a medium containing glucose (MGlu-repressive conditions, negative control) or galactose (MGal-induced conditions) as carbon sources. In order to test cell proliferation, cells expressing APOL1 variants and control cells were plated on MGlu and MGal. No significant differences were observed in the number of colonies between cells expressing or not expressing APOL1 (Fig. 1B). As expected, no significant difference was observed between the different cells on MGlu, the repressive medium. Similarly, Fig. 1C shows no significant differences in the proliferation of cells expressing or not expressing an APOL1 variant on liquid MGal or MGlu media although its expression was properly induced in the MGal medium (Fig. 1A). Thus, APOL1 expression levels do not influence cell proliferation in these conditions. In the presence of glucose or galactose, yeast cells primarily undergo anaerobic fermentation, generating ROS. In order to monitor ROS generation in induced and non-induced conditions, cells were stained with DHE detecting superoxide molecules and analyzed by flow cytometry in the present study. As presented in Fig. 1D and quantified in Fig. 1E, this analysis did not detect any significant difference in the fraction of the cell population producing ROS between the strains expressing or not APOL1.

In the presence of a fermentable carbon source such as glucose or galactose, yeast cells preferentially generate energy by undergoing fermentation, and can grow normally with a minimal level of mitochondrial respiration, generating ethanol as the end product of fermentation. When fermentable carbon sources become limiting, genes required for respiration are induced, and ATP is generated by metabolizing non-fermentable carbon sources such as glycerol, ethanol or lactate in the mitochondria (17).

In order to determine whether human APOL1 could interfere with mitochondrial function, cells initially grown on MGal to induce APOL1 expression were transferred to an MGly medium, containing glycerol, a non-fermentable carbon source. As presented in Fig. 2A, yeast proliferation was inhibited in MGly media when APOL1 was induced (Fig. 2B) compared with the control culture. This indicated that APOL1 was able to inhibit yeast proliferation in conditions in which aerobic respiration is required, indicating that this protein could interfere with mitochondrial function.

As mitochondria are primarily involved in respiration, the present study assessed the IRC in cells expressing or not APOL1 variants. This index measures the percentage of viable respiration-competent cells and reflects the fraction of cells that can grow on non-fermentable (glycerol) carbon sources compared with the fraction of cells growing on fermentable (galactose) carbon sources (18). At the same time, the expression levels of the induced gene were verified (Fig. 2D). As presented in Fig. 2C, while the IRC value was 100% at the beginning of the experiment for all three strains, the IRC value started decreasing at day 1 only in the cells expressing APOL1 and APOL1 ∆BH3. This decrease continued, reaching almost 20% after 7 days, while IRC remained higher than 70% for the control strain. The absence of the BH3 domain in APOL1 ∆BH3 expressing cells had no effect on the number of viable respiration-competent cells.

As a result of the cell proliferation inhibition observed on MGly media and the drop in respiratory index, the present study investigated whether the mitochondrial membrane potential (∆ψm) of yeast is affected by APOL1 induction. Maintenance of the membrane potential is key for mitochondrial functions (19). Thus, cells expressing or not expressing native APOL1 or APOL1-∆BH3 on a fermentable carbon source (MGal), were incubated with TMRE, a chemical dye able to accumulate in the mitochondria depending on membrane polarization (20). The positive control, which included treatment with FCCP (an ionophore that uncouples the electron transport chain from ATP production), completely prevented TMRE staining. As presented in Fig. 3, cells expressing APOL1, whether native or ∆BH3, showed a loss of membrane potential. TMRE uptake into the mitochondria was decreased by 68 and 70% in cells expressing native APOL1and ∆BH3 APOL1 respectively (P<0.0001). This suggests that APOL1 expression levels promote loss of mitochondrial membrane potential and that this ability does not require the BH3 domain.

As APOL1 induction provoked mitochondrial membrane depolarization, the present study investigated a potential direct APOL1 interaction within the yeast mitochondria. In order to assess the subcellular localization of APOL1 in yeast, APOL1-expressing cells were grown on galactose medium for 24 h and mitochondria enrichment was performed using these cells. A fraction of HA-APOL1 was consistently found in the mitochondrial-enriched fraction (enriched mitochondria) as validated by the detection of the outer membrane mitochondrial channel-forming protein porin in this fraction (Fig. 4).

As APOL1 localizes in the mitochondria and interferes with proper function, the present study investigated whether their morphology was also affected. Morphology was visualized using mt-GFP, a mitochondria-targeted GFP protein. Cells co-expressing native APOL1 or APOL1-∆BH3 with mt-GFP were grown in MGal. In control cells not expressing APOL1, a normal branched tubular mitochondrial network located below the cell cortex (21) was observed. In contrast, in yeast cells expressing APOL1, the mitochondrial network was altered; the branched network disappeared and fluorescent patches appeared predominantly at the periphery of some of the cells; the mitochondrial signal in APOL1-expressing cells concentrated in fewer mitochondria that were larger compared with those in control cells and mt-GFP was not detectable in certain APOL1-expressing cells (Fig. 5A).

In order to test if this morphological change was accompanied by a change in mitochondrial content, cells were grown on non-inducible and non-repressive medium (raffinose) and APOL1 was induced in a time-dependent manner by the addition of galactose to the growth medium. Using western blotting, the present study observed the stability of two mitochondrial components after inducing APOL1 for 4 h in the cells. Fig. 5C shows that HA-APOL1 (used as a control) was immunodetectable during the 4 h of induction. Porin, a mitochondrial outer membrane protein, remained stable during the 4 h induction. A decrease in the mitochondrial mt-GFP signal (35 KDa) was observed.

In order to further analyze these different structural patterns, the present study used a fluorescence microscopy assay during APOL1 induction kinetics. Cells were grown in MRaf and galactose was added at time (T)=0 to induce HA-APOL1 expression. At different times after the induction, cell aliquots were collected for mitochondrial visualization using mt-GFP as a marker and for immunoblot analysis. The present study identified different categories of the mitochondrial morphological structures that are consistent with what has been previously described (22). At T=0, 92% of the cells contained the typical tubular shaped mitochondria (Fig. 6 and Table II). Mitochondria remained tubular in 90% of the cells at (T)=0. Following 1 h of APOL1 induction, 67.2% still contained a normal mitochondrial structure while 5% had fragmented but widespread mitochondria, 12.9% showed mitochondria aggregated to one side of the cell, and 14.6% of cells lost the mitochondrial signal. In the control cells, mitochondria remained tubular throughout the experiment, while only 26.0% of APOL1-expressing cells displayed normal tubular mitochondria following 4 h of induction. At T=4, a notable proportion of the cell population lacked detectable mitochondrial structure (42.5%). This suggested that prolonged APOL1 expression levels may progressively modify mitochondrial structure.

As APOL1 uptake by African trypanosomes promotes lysosome swelling (23), the present study also observed the behavior of lysosome-like organelles, namely the vacuole, following APOL1 induction in yeast cells. Yeast cells were grown on MGal medium and stained with the fluorescent dye FM4-64, which accumulated at the vacuolar membrane. As presented in Fig. 7, APOL1-expressing cells exhibited alterations in vacuolar morphology, showing a vacuole fragmented into smaller vesicles compared with the control cells, which possessed a single, regular and large vacuole. These smaller vesicles restained as intensely as the single wild type vacuole.