APOL1 Kidney Risk Variants Induce Cell Death Via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore

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APOL1 Kidney Risk Variants Induce Cell Death via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore

Shrijal S. Shah, Herbert Lannon , Leny Dias, Jia-Yue Zhang, Seth L. Alper, Martin R. Pollak, and David J. Friedman

Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

ABSTRACT Background Genetic Variants in Apolipoprotein L1 (APOL1) are associated with large increases in CKD rates among African Americans. Experiments in cell and mouse models suggest that these risk-related polymorphisms are toxic gain-of-function variants that cause kidney dysfunction, following a recessive mode of inheritance. Recent data in trypanosomes and in human cells indicate that such variants may cause toxicity through their effects on mitochondria. Methods To examine the molecular mechanisms underlying APOL1 risk variant–induced mitochondrial dysfunction, we generated tetracycline-inducible HEK293 T-REx cells stably expressing the APOL1 nonrisk G0 variant or APOL1 risk variants. Using these cells, we mapped the molecular pathway from mitochondrial import of APOL1 protein to APOL1-induced cell death with small interfering RNA knockdowns, pharmacologic inhibitors, blue native PAGE, mass spectrometry, and assessment of mitochondrial permeability transition pore function. Results We found that the APOL1 G0 and risk variant proteins shared the same import pathway into the mitochondrial matrix. Once inside, G0 remained monomeric, whereas risk variant proteins were prone to forming higher-order oligomers. Both nonrisk G0 and risk variant proteins bound components of the mitochondrial permeability transition pore, but only risk variant proteins activated pore opening. Blocking mitochondrial import of APOL1 risk variants largely eliminated oligomer formation and also rescued toxicity. Conclusions Our study illuminates important differences in the molecular behavior of APOL1 nonrisk and risk variants, and our observations suggest a mechanism that may explain the very different functional effects of these variants, despite the lack of consistently observed differences in trafﬁcking patterns, intracellular localization, or binding partners. Variant-dependent differences in oligomerization pattern may underlie APOL1’s recessive, gain-of-function biology.

JASN 30: 2355–2368, 2019. doi: https://doi.org/10.1681/ASN.2019020114

African Americans develop ESKD much more fre- quently that other groups. A large fraction of this disparity is due to genetic variants in the APOL1 Received February 5, 2019. Accepted August 15, 2019. gene. Inheriting two copies of APOL1 risk variants Published online ahead of print. Publication date available at (RVs), known as G1 and G2, causes high rates of www.jasn.org.

FSGS, HIV-associated nephropathy and hypertension- Correspondence: Dr. David J. Friedman, Beth Israel Deaconess associated ESKD.1–4 Medical Center, 330 Brookline Avenue, Boston, MA 02215. Of the six members of the APOL gene family, Email: [email protected] APOL1 is the sole member with a signal peptide Copyright © 2019 by the American Society of Nephrology

JASN 30: 2355–2368, 2019 ISSN : 1046-6673/3012-2355 2355 BASIC RESEARCH www.jasn.org permitting cellular export.5,6 Circulating non-risk APOL1 Significance Statement (G0) confers resistance to trypanosome Trypanosoma brucei brucei through ion channel formation and trypanolysis, but Some variants in APOL1 are associated with high CKD rates in cannot protect against the two subspecies Trypanosoma brucei African Americans, but the molecular mechanism of disease re- rhodesiense and Trypanosoma brucei gambiense.7,8 These highly mains elusive. Previous studies demonstrated that expression of APOL1 risk variants is associated with mitochondrial dysfunction. In pathogenic subspecies have evolved multiple mechanisms to this study, the authors show that import of APOL1 protein into inactivate APOL1-mediated lysis.9,10 G1 (two amino acid sub- mitochondria is essential for risk variant–mediated cytotoxicity, and stitutions: S342G and I384M) and G2 (a two-amino-acid de- map the APOL1 import pathway. They found that whereas APOL1 is letion: del388N389Y) mutations originated in sub-Saharan mostly monomeric, risk variant APOL1 can form large oligomers Africa and rose to high frequency because they provided a and cause opening of the mitochondrial permeability transition pore, ultimately leading to cell death. This difference in propensity selective advantage in regions with pathogenic trypano- of different variants to oligomerize could help explain APOL1 risk – somes.1,11 13 Although inheriting a single copy of G1 or G2 variants’ gain-of-function biology despite a recessive mode of in- is sufficient to enhance protection against resistant trypano- heritance. Understanding APOL1 trafficking and interactions could somes, inheriting two copies increases risk of developing kid- help inform new therapeutic approaches. ney disease. Not all individuals with two copies of the RVs develop kid- could explain why multiple modes of cell death have been ney disease, suggesting a second hit is required for disease to observed in association with APOL1 overexpression. develop in high-risk individuals. APOL1 RVs increase risk in a recessive manner, but unlike most variants that cause a recessive mode of inheritance, evidence suggests that APOL1 RVs METHODS are toxic, gain-of-function mutations.14 This hypothesis is supported by the fact that most mammals and even some Cells and Antibodies primates have no APOL1 gene, and at least one human is HEK293 cells expressing tetracycline-inducible APOL1 inte- completely null for APOL1 while having no apparent kidney grated at a single locus were generated using the T-REx system dysfunction.6,15 Although the increased risk of kidney disease (Thermo Fisher Scientific). APOL1 sequences used for each due to APOL1 RVs has been well established, it remains un- genotype are shown in Supplemental Table 1. Cells were cul- certain how the APOL1 RVs promote kidney injury. tured at 37°C with 5% CO2 in DMEM (Corning) supple- Our goal was to address several important questions about mented with 10% tetracycline-free FBS (Atlanta Biologicals). APOL1 biology. First, the nature of APOL1 gain-of-function Cells were validated to be free of any Mycoplasma con- toxicity has remained elusive despite many well designed tamination. Anti-APOL1 rabbit polyclonal antibody studies. Evidence supporting many different cell death (HPA018885, lot #E114503) and anti-FLAG (F1804) — mechanisms apoptosis, autophagy, necrosis, pyroptosis, mouse monoclonal antibody were from Sigma. Anti- — 16–32 and necroptosis has been put forth. Second, the reason ACSL4 (SC-365230), anti-TOMM20 (SC-17764), anti- why two RVs are required for kidney disease, if the variants TOMM22 (SC-101286), anti-TOMM70 (SC-390545), are truly gain-of-function mutations, is not yet understood. anti-TIMM23 (SC-514463), anti-TIMM17 (SC-271152), The recessive mode of inheritance rather than an additive anti-HSPA9 (SC-133137), anti-HSP60 (SC-13115), anti- effect of APOL1 RVs has been proposed to involve oligomer ATP5A (SC-136178), anti-ATP5B (SC-55597) mouse mAb, formation or threshold effects, but there is little direct evi- and anti-APOL1 (SC-18759) goat polyclonal antibodies 33 fi denceforthesetheoriestodate. Third, de nitive differ- were from Santa Cruz Biotechnology. Anti-TIMM22 ences in behavior between G0 and RV APOL1 relating to (14927–1-AP) rabbit polyclonal antibody was from Protein- fi subcellular localization and/or af nity for binding partners tech. Anti-SLC25A5 (14671S) anti-calreticulin (12338S), have not been consistently demonstrated.34,35 We explore a anti-GM130 (12480S), anti-Rab7 (9367S), anti-LC3 modelthatmaybegintounifythesequestions. (12741S), anti-myc (2278S) rabbit polyclonal antibodies In this study, we build on the observation that APOL1 RVs were from Cell Signaling Technologies. cause mitochondrial dysfunction27,32,36 by (1) mapping an APOL1 mitochondrial import pathway, (2) demonstrating en- Preparation of Mitochondria-Enriched Fractions hanced APOL1 RV self-aggregation that occurs after transport Mitochondrial enrichment protocol was adapted from to the mitochondria, (3)defining a set of inner mitochondrial Jastroch et al.37 Briefly, cells were scraped in PBS and centri- membrane (IMM) binding partners of APOL1 that include fuged at 5003g for 5 minutes. Cell pellets were resuspended in several major mitochondrial permeability transition pore STE medium (250 mM sucrose, 5 mM Tris, 2 mM EGTA; pH 7.4) (mPTP) constituents and modulators, and (4) activating the and plasma membranes were disrupted using a dounce ho- mPTP, triggering first mitochondrial dysfunction and then mogenizer. The whole homogenate was centrifuged for 10 cell death. Our data on differential oligomer formation may minutes at 10003g and the supernatant was saved in a fresh ultimately help illuminate why both alleles must be RVs for tube. To increase yields, the cell pellet was resuspended again disease to occur, and our findings regarding mPTP activation in STE, dounced, and centrifuged at 10003g for 10 minutes.

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The two supernatants were pooled and filtered through a cells were pretreated with respective dose of drug for 2 hours 5 mMsyringefilter. An aliquot of filtered lysate was saved as and then induced with 50 ng/ml tetracycline for 18 hours. loading control and denoted on figures as “total” protein. The Cytotoxicity/viability was measured using the MultiTox- remaining lysate was spun at 10,0003g for 10 minutes at 4°C. Fluor Multiplex Cytotoxicity Assay kit (Promega). The “supernatant/cytosolic” fraction was transferred to a fresh tube. The resulting mitochondria-enriched pellet was washed Blue Native PAGE and SDS-PAGE twice and then resuspended in STE medium. For blue native PAGE, cell pellets were solubilized with 50 mM imidazole/HCl (pH 7), 50 mM NaCl, 1% digitonin lysis buffer Proteinase K Protection Assay supplemented with Protease Inhibitor Cocktail (Sigma), and The protocol was adapted from Ryan et al.38 Mitochondrial- PhosSTOP tablets (Sigma). After 30 minutes of incubation, enriched pellet obtained after differential centrifugation was lysates were centrifuged at 15,0003g for 10 minutes at 4°C resuspended in STE buffer or hypotonic solution (5 mM Tris, and cell pellets were discarded. For blue native PAGE and pH 7.4) and divided into three. Samples were incubated on ice SDS-PAGE after differential centrifugation, each fraction with only STE or 25 mg/ml Proteinase K or 25 mg/ml Proteinase was solubilized in 1% digitonin in STE buffer. Protein con- Kwith 0.1% Triton X-100 for 1 hour; 5 mM PMSF was added to centrations were measured using DC Assay Kit (Bio-Rad). For stop the reaction. After 10 minutes incubation on ice, samples blue native PAGE, 43 NativePAGE Sample Buffer and 5% were boiled in SDS loading buffer for immunoblotting. G-250 Additive (final 0.25%) were added to the lysates and were run on 3%–12% Bis-Tris protein gels (Thermo Fisher Small Interfering RNA Transfections Scientific). For SDS-PAGE, cells were lysed in RIPA buffer and Silencer select small interfering RNA (siRNA) (Ambion) the lysate centrifuged at 17,0003g. Supernatants were boiled were used at a final concentration of 40 nM with RNAiMAX in reducing SDS sample buffer at 95°C for 5 minutes, sepa- (Invitrogen) for knockdown experiments. All experiments rated on 4%–20% Criterion TGX gels, and then transferred were performed via reverse transfections (siRNA/OptiMEM/ onto PVDF membranes (Millipore). After blocking, mem- RNAiMAX mix was added to each well before overlaying branes were incubated overnight in primary antibody, washed, the cells). siRNAs used were: siNT (Negative Control #2), incubated for 1 hour in HRP-linked secondary antibody, and TOMM20 (s18950), TOMM22 (s32550), TOMM70 washed again. Blots were imaged with film after brief incuba- (s19107), TIMM22 (s26725), TIMM23 (s223113), TIMM17 tion in SuperSignal HRP substrate solution (Thermo Fisher (s20424, s20425), HSPA9 (s6989, s6990), SLC25A3 (s10428), Scientific). SLC25A4 (s223817), SLC25A5 (s1375), CYPD (s19662), and APOL1 (s16255). Preornithine Transcarbamylase Mitochondrial Import Assay Mitochondria Functional (Seahorse) Assay Flag-tagged human ornithine transcarbamylase precursor HEK293 empty vector (EV), G0, G1, and G2 cells were re- plasmid (NM_000531; Origene) was in vitro transcribed verse transfected with 40 nM Non-Target or TOMM20 and translated in vitro (TNT quick coupled transcription/ siRNA in a V3-PS cell culture plate (Agilent Technologies) translation system, L1170; Promega). Mitochondria-enriched for 48 hours. Cells were then induced with 50 ng/ml tetracy- pellets isolated from HEK293 EV,G0, G1, and G2 cells induced cline for 8 hours. Media was replaced with Agilent Seahorse with 50 ng/ml tetracycline for 6 hours were resuspended in XF base medium supplemented with 10 mM glucose, 1 mM import buffer: 0.1% (w/v) BSA, 250 mM sucrose, 80 mM KCl, sodium pyruvate, and 2 mM L-glutamine, at pH 7.4. Oxygen 5mMMgCl2,2mMKH2PO4,and10mMMOPS-KOH,at consumption rate was measured using the XFe96 Extracellu- pH 7.4. Final import reactions contained 2 mM ATP, 2 mM lar Flux Analyzer (Agilent Technologies). Next, 1 mMoligomy- NADH, 5 mM succinate, mitochondria, and 3% (v/v) in cin, 0.25 mM FCCP,or 0.5 mM mix of antimycin A and rotenone vitro translated preornithine transcarbamylase (pOTC). pOTC were serially injected to measure ATP production, maximal re- import was performed at 30°C for 30 minutes. Mitochondria spiratory capacity, and nonmitochondrial respiration, respec- were reisolated by spinning at 10,0003g for 10 minutes before tively. After obtaining all measurements, media was aspirated subjecting to SDS-PAGE. and protein concentrations were measured using a bicincho- ninic acid (BCA) assay. Oxygen consumption rate was normal- Immunoprecipitation Mass Spectrometry ized to protein concentration in each well. HEK293 EV, G0, G1, and G2 cells were induced with 50 ng/ml tetracycline for 8 hours. Cells were lysed in 50 mM Tris/HCL Cytotoxicity Assays (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 0.3% CHAPS lysis For siRNA knockdown experiments cells were reverse trans- buffer supplemented with Protease Inhibitor Cocktail and fected with 40 nM of respective siRNA in a 96-well, black- PhosSTOP tablets. Lysates were incubated overnight with walled, clear-bottomed plate, allowed to incubate for 48 hours, anti-APOL1 goat polyclonal antibody crosslinked Protein and then induced with 50 ng/ml tetracycline for 18 hours. G Dynabeads. Beads were washed three times with ice-cold For cyclosporin A (CsA) and NIM811 rescue experiments, lysis buffer and proteins were eluted with nonreducing

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Laemmli sample buffer. Samples were separated on a 10% mitochondria-enriched fractions. TOMM20 was used as an Criterion gel. After gel staining with colloidal blue (Thermo OMM marker, TIMM23 as an inner mitochondrial membrane Fisher Scientific), the gel was cut into several small bands (IMM) marker, HSP60 as a mitochondrial matrix marker, and and the gel fragments sent for protein identification by ACSL4 as a marker of mitochondria-associated endoplas- liquid chromatography with tandem mass spectrometry mic reticulum membrane (MAM). Proteinase K treatment (LC/MS/MS). The mitochondrial protein list was generated fully degraded MAM and OMM proteins but most APOL1 by performing gene ontology enrichment analysis followed remained protected (Figure 1B), suggesting that APOL1 was by manual curation using UniProtKB data. A minimum of present either in the IMM, the intermembrane space, or five total peptides cutoff was applied after subtracting back- within the mitochondrial matrix.38 To refine APOL1 localiza- ground from EV lanes. tion, mitochondria-enriched fraction was resuspended in hypotonic medium to disrupt the OMM, then subjected to Proteinase Live Cell Imaging K treatment to digest IMM-spanning proteins.38 Hypotonicity HEK293 EV, G0, G1, and G2 cells were induced with 50 ng/ml followed by Proteinase K digestion degraded MAM, tetracycline for 6 hours. mPTP opening was assessed using the OMM, and IMM proteins, whereas HSP60 and APOL1 re- Image-IT live mitochondrial transition pore assay kit (Thermo mained protected (Figure 1C), indicating that a fraction of Fisher Scientific). Briefly, cells were stained with 1 mMcalcein APOL1 is translocated into the mitochondrial matrix despite acetoxymethyl ester (calcein-AM), 200 nM Mitotracker Red its lack of a canonical N-terminal mitochondrial matrix tar- CMXRos, 1 mM Hoechst 3334264mMCoCl2 for 15 minutes. geting signal sequence, on the basis of sequence prediction Images were then acquired as z-stacks. The raw integrated density programs.40 The purity of the mitochondria-enriched frac- of each optical slice was added and then divided by the number of tion was immunoblot-validated by probing for markers of cells in that field, using ImageJ. Mitochondrial membrane po- endoplasmic reticulum, Golgi, plasma membrane, endo- tential was measured in cells were stained for 30 minutes with somes, and autophagosomes (Supplemental Figure 1). 50 nM tetramethylrhodamine (TMRM; Invitrogen). All images were acquired with the Zeiss LSM live-cell confocal system. Specific OMM and IMM Proteins Are Required for APOL1 Translocation to Mitochondrial Matrix and for Statistical Analyses APOL1-Induced Cytotoxicity All data shown are presented as means6SEM unless stated Multiple trafficking pathways have been defined for import otherwise. Data sets were analyzed for statistical significance of mitochondrial proteins.41,42 Most matrix-targeted proteins by ANOVAwith the Dunnett post-test, using GraphPad Prism. contain a presequence that is sequentially recognized by TOMM20, TOMM22, and TOMM40, and then directed to the matrix via the TIMM23/TIMM17 complex. To test RESULTS whether TOMM20 was essential for APOL1 localization, we knocked down TOMM20 in stably inducible APOL1-expressing G0 and RV APOL1 Translocate to the Mitochondrial HEK293 cells, then induced APOL1 expression. Six hours Matrix after induction, mitochondria were isolated by differential cen- APOL1 RVs have been shown to impair maximum respiratory trifugation as above, then subjected to Proteinase K treatment. capacity, whereas G0 either had no effect or enhanced mito- Eliminating TOMM20 rendered APOL1 susceptible to Pro- chondrial respiratory function.27,32 APOL1 protein has been teinase K degradation (Figure 2, A and B), suggesting APOL1 shown to localize to mitochondria by immunostaining in several was no longer protected inside the matrix. We also tried doing studies, but it is unclear from imaging alone whether APOL1 gets the same experiment after knocking down TOMM70, an es- imported into mitochondria or is instead in close proximity sential component of carrier pathway for protein insertion (e.g., apposed to the outer mitochondrial membrane [OMM] into IMM but not matrix import. In contrast, elimination or located in mitochondrial-associated membranes).27,32,39 We of TOMM70 did not sensitize APOL1 to Proteinase K diges- sought biochemical confirmation of APOL1 mitochondrial lo- tion (Supplemental Figure 2). calization and identification of the mechanism of APOL1 traffick- We next asked if blocking APOL1 translocation to the mi- ing into mitochondria. HEK293 cells stably transfected with tochondria via TOMM20 knockdown could reverse APOL1 APOL1 (G0 or RVs) were induced with 50 ng/ml tetracycline for effects on mitochondrial metabolism. Seahorse analysis on 6 hours and then fractionated using a differential centrifugation cells in which TOMM20 was knocked down showed partial protocol.37 Total cell lysates, cytosolic fraction, and mitochondrial- rescue of basal and maximum respiration capacity in G1 enriched fraction were separated by SDS-PAGE. Tubulin served and G2 cells (Supplemental Figure 3). We also asked whether as a cytosolic marker and TOMM20 as a mitochondrial marker. knocking down various components of the inner and outer APOL1 G0, G1, and G2 each cofractionated with the TOMM20- mitochondrial translocase machinery could reduce G1 and G2 positive, mitochondria-enriched fraction (Figure 1A). cytotoxicity. Knockdown of TOMM20 or TOMM22 rescued To determine the submitochondrial localization cell death associated with induction of G1 or G2, but knock- of APOL1, we performed Proteinase K protection assays on down of TOMM70 did not rescue cell death (Figure 2C).

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A EV G0 G1 G2

Total Sup Mito Total Sup Mito Total Sup Mito Total Sup Mito 50 APOL1 37

Tubulin 50

TOMM20 15

B EV G0 G1 G2 Proteinase K - + +-+ +-+ +-+ + TritonX-100 - - +-- +-- +-- + 50 APOL1 37

TOMM20 15 25 TIM23 20

HSP60 50 100 ACSL4 75

C EV G0 G1 G2 Hypotonic Soln +++ +++ +++ +++ Proteinase K -++ -++ -++ -++ TritonX-100 --+ --+ --+ --+ 50 APOL1 37

TOMM20 15 25 TIM23 20

HSP60 50 100 ACSL4 75

Figure 1. APOL1 translocates into the mitochondrial matrix. (A) APOL1 cofractionates with the mitochondrial marker TOMM20, indicating mitochondrial association. HEK293 cells stably transfected with APOL1 (EV, G0, G1, or G2) were induced with tetracycline for 6 hours. The cell lysate was separated by differential centrifugation into a mitochondria-enriched fraction (TOMM20 positive) and a supernatant fraction (tubulin positive). (B) APOL1 is translocated into mitochondria. Isolated mitochondria were subjected to Proteinase K digestion to degrade proteins bound to the OMM and MAM-associated proteins. Proteinase K digests the OMM protein TOMM20 and the MAM protein ACSL4 but not the IMM protein TIM23 or matrix protein HSP60. Continued presence of APOL1 after protease digestion indicates prior mitochondrial internalization. (C) APOL1 translocates to mitochondrial matrix. Isolated mitochondria were ﬁrst incubated in hypotonic solution to lyse the OMM, then subjected to Proteinase K digestion. OMM (TOMM20), IMM (TIM23), and MAM (ACSL4) proteins were digested, but matrix proteins HSP60 and APOL1 are protected. Addition of TritonX-100 digestion solubilized lipids of the IMM and rendered APOL1 in the mitochondrial matrix susceptible to digestion by Proteinase K.

Similarly, knockdown of key components of the IMM and TIMM17, and HSPA9) all rescued cell death associated with matrix machinery required for most matrix translocation of induction of G1 or G2. Conversely, knockdown of TIMM22, an proteins with classic mitochondrial presequences (TIMM23, essential component of the carrier pathway for IMM-directed

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A G0 G1 G2 B siNT siTOMM20 siNT siTOMM20 siNT siTOMM20 Proteinase K -++-++ -++-++ -++-++ TritonX-100 --+--+ --+--+ --+--+ APOL1 Densitometry (proteinase K treated/untreated) APOL1 50 37 0.6 siNT TOMM20 15 siTOMM20 0.4 25 TIM23 20 0.2 HSP60 50 100 0.0 ACSL4 75 G0 G1 G2

C 0.20 EV 0.5 G0

0.4 0.15 0.3 0.10 0.2 0.05 0.1 Cytotoxicity/Viability Cytotoxicity/Viability

0.00 0.0

siNT siNT siAPOL1 siAPOL1 siTOMM20siTOMM22siTOMM70 siTOMM20siTOMM22siTOMM70

1.2 G1 1.4 G2

1.0 1.2 1.0 0.8 0.8 0.6 0.6 0.4 ** 0.4 ** ** Cytotoxicity/Viability Cytotoxicity/Viability ** 0.2 0.2 ** ** 0.0 0.0

siNT siNT siAPOL1 siAPOL1 siTOMM20siTOMM22siTOMM70 siTOMM20siTOMM22siTOMM70

D 0.20 EV 0.5 G0

0.4 0.15 0.3 0.10 0.2 0.05 0.1 Cytotoxicity/Viability Cytotoxicity/Viability ** 0.00 0.0

siNT siNT siApol1 siApol1 siTIMM23 siTIMM22 siTIMM23 siTIMM22 siTIMM17siTIMM17 #1siHSPA9 #2siHSPA9 #1 #2 siTIMM17siTIMM17 #1siHSPA9 #2siHSPA9 #1 #2

1.2 G1 1.0 G2

1.0 0.8 0.8 0.6 0.6 ** ** 0.4 0.4 ** ** ** ** ** ** 0.2 ** 0.2 Cytotoxicity/Viability ** Cytotoxicity/Viability ** ** 0.0 0.0

siNT siNT siApol1 siApol1 siTIMM23 siTIMM22 siTIMM23 siTIMM22 siTIMM17siTIMM17 #1siHSPA9 #2siHSPA9 #1 #2 siTIMM17siTIMM17 #1siHSPA9 #2siHSPA9 #1 #2

Figure 2. APOL1 translocation to mitochondrial matrix is dependent on IMM and OMM translocase machinery. (A and B) Knockdown of TOMM20 reduces mitochondrial APOL1. Nontarget siRNA (siNT) does not affect APOL1 translocation, as demonstrated by per- sistence of intact APOL1 in the presence of Proteinase K. Knockdown of TOMM20 (siTOMM20) substantially decreased mitochondrial APOL1 content after Proteinase K treatment. (C) Knockdown of TOMM20 and TOMM22 in matrix protein translocation pathway

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A B Total Mitochondria Supernatant EV G0 G1 G2 EV G0 G1 G2 EV G0 G1 G2 EV G0 G1 G2

1236 1236 1048 1048

APOL1 APOL1 720 720

480 480 Native Blot Native Blot 242 242 146 146 66 66

50 APOL1 50 37 APOL1

37 SDS Blot

SDS Blot TOMM20 15

Figure 3. APOL1 RVs form higher-ordered oligomers that are enriched in the mitochondria. (A) Soluble fractions from digitonin lysates of APOL1-expressing cells were subjected to native PAGE, demonstrating that RV APOL1 forms a range of higher-ordered oligomers. (B) Differential centrifugation with mitochondrial isolation followed by digitonin lysis and native PAGE demonstrate that higher-order RV APOL1 is present primarily in the mitochondrial enriched fraction. Discrete oligomers appear at up to 1.2 Md; larger aggregates are retained in the well at top. (A and B) Stably transfected HEK293 cells expressing APOL1 were induced for 15 hours with (A) 25 ng/ml tetracycline or (B) 6 hours with 50 ng/ml tetracycline before lysis. proteins, had no effect on APOL1 RV–associated cell death G1- and G2-induced toxicity, we examined the cytosolic ver- (Figure 2D; see Supplemental Figure 4 for validation of siRNA sus mitochondrial-enriched fractions on native gels. Most of knockdown efﬁciencies). Our data suggest that mitochondrial the G1 and G2 higher-ordered oligomers were located in the matrix delivery of APOL1 is needed for APOL1-induced mitochondria-enriched fraction (Figure 3B). cytotoxicity. To distinguish whether G1- and G2-containing oligomers form before or after mitochondrial translocation, cells G1 and G2 Form Oligomers after Translocation to the knocked down for TOMM20 were induced and subjected to Mitochondrial Matrix blue native PAGE (Supplemental Figure 6A). There was a Investigators have hypothesized that APOL1 multimerization markedreductionintheRVAPOL1oligomerizationaf- may be central to the explanation of why two risk alleles are ter TOMM20 knockdown, indicating that oligomers are required to increase kidney disease risk.33 We documented by forming inside the mitochondria. Knockdown of the carrier coexpressing Myc- and FLAG-tagged APOL1 constructs in pathway protein TOMM70 had no effect on multimerization HEK293 cells, followed by pulldown with FLAG beads and (Supplemental Figure 6B), suggesting the destination of immunoblot for Myc, that APOL1 undergoes homomeriza- APOL1 monomers was not directly to the IMM but rather tion (Supplemental Figure 5). We tested APOL1 behavior in the matrix via the TOMM20-dependent pathway. To conﬁrm our stable APOL1-expressing cell lines using nonreducing, translocation to the mitochondrial matrix was necessary for nondenaturing blue native PAGE. We found that G1 and G2 oligomerization, native gels were run on cell lysates after knock- formed a range of higher-ordered oligomers, whereas G0 ap- ing down TIMM17 (Figure 4A) and HSPA9 (Figure 4B), pro- peared predominantly monomeric (Figure 3A). Because teins required for both APOL1 matrix translocation and cyto- APOL1 translocation to the mitochondria is important for toxicity. Blocking APOL1 translocation into the mitochondrial

reduces RV APOL1-induced cell death. In contrast, knockdown of TOMM70, an OMM protein involved in targeting proteins to the IMM in the carrier pathway, does not affect RV APOL1-induced cell death. TOMM70 knockdown efﬁciency is shown in Supplemental Figure 6B. (D) Knockdown of TIMM17 and TIMM23 in the matrix protein translocation pathway and knockdown of mitochondrial matrix translocation chaperone HSPA9 independently reduced APOL1-induced cell death. In contrast, knockdown of the carrier pathway protein TIMM22 that shuttles incoming proteins into the IMM does not affect APOL1-induced cell death. TIMM22 knockdown efﬁciency is shown in Supple- mental Figure 6C. (A–D) Stably transfected HEK293 cells were treated with 50 ng/ml of tetracycline for 6 (A and B) or 18 (C and D) hours. (B) Densitometry was performed on three independent experiments. (C and D) Each dot represents an independent experiment. **P,0.001 comparing each siRNA to siNT-treated cells.

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A B

EV siNT#2EV siHSPA9EV siHSPA9G0 #1 siNT#2G0 #2 siHSPA9G0 siHSPA9G1 #1 siNT#2G1 #2 siHSPA9G1 siHSPA9G2 #1 siNT#2G2 #2 siHSPA9G2 siHSPA9 #1 #2 EV siNT#2EV siTIMM17EV siTIMM17G0 siNT#2#1G0 siTIMM17 #2G0 siTIMM17G1 siNT#2#1G1 siTIMM17#2G1 siTIMM17G2 #1siNT#2G2 siTIMM17#2G2 siTIMM17 #1 #2

1236 1236 1048 1048

720 APOL1 720 APOL1 480 480 Native Blot Native Blot 242 242 146 146 66 66

50 50 APOL1 APOL1 37 37 100 20 SDS Blot TIMM17 SDS Blot HSPA9 75 15

Figure 4. APOL1 transport to mitochondrial matrix is required for formation of higher-order oligomers. (A and B) Knockdown of TIMM17 (a key component of the IMM protein translocation machinery) or HSPA9 (a mitochondrial matrix translocation chaperone needed to guide proteins through the translocation machinery) prevents APOL1 higher-order oligomer formation (in addition to preventing cell death: see Figure 2D). Conversely, knockdown of TIMM22 affects neither cell death nor APOL1 oligomer formation (see Supplemental Figure 6C). Stable APOL1-transfected cells were treated for 48 hours with siRNA prior to inducing APOL1 with 50 ng/ml tetracycline. Cells were lysed with digitonin after 15 hours and were fractionated by native PAGE (large top panels) or SDS page (lower narrow panels). The signal from the monomeric fraction is saturated in these images. matrix largely prevented G1 and G2 oligomerization, again APOL1-binding partners. We observed that APOL1 pulled suggesting oligomerization was occurring inside the mito- down nearly all putative components or modulators of the chondria. Conversely, knockdown of TIMM22 (responsible mPTP, an IMM complex that forms a pore between the matrix for import of proteins directly into the IMM) that does and intermembrane space, and several mPTP compo- not alter APOL1 RV–associated cytotoxicity (Figure 2D) also nents were among the most enriched mitochondrial proteins failed to inhibit G1 or G2 oligomerization (Supplemental (Table 1; full list in Supplemental Table 2).43–45 We validated Figure 6C). These data show that G1 and G2 oligomerization several of these candidate proteins by immunoprecipitating occurs after translocation into mitochondrial matrix, and that with antibodies to ATP5A, ATP5B, and SLC25A5/ANT2, and blocking mitochondrial import of APOL1 RVs prevents both then immunoblotting with anti-APOL1 antibody. We found APOL1 oligomerization and cell death. that the association of these mitochondrial proteins with RVs We tested whether APOL1 import into mitochondria could generally exceeded their association with G0 (Supplemental be indirectly causing mitochondrial dysfunction by blocking Figure 8). import of other mitochondrial proteins. We performed an To test whether APOL1 expression leads to mPTP opening, 46 import assay using in vitro translated pOTC and mitochondria we used a CoCl2-calcein fluorescence quenching assay. Non- isolated from EV, G0-, G1-, and G2-expressing cells. pOTC fluorescent calcein-AM freely diffuses throughout the cells. has a mitochondrial matrix targeting presequence, which is The acetoxymethyl ester group is then cleaved by intracellular cleaved by mitochondrial peptidases after import, producing proteases, yielding hydrophilic, fluorescent calcein. CoCl2, mature ornithine transcarbamylase. pOTC cleavage was ob- which can quench calcein fluorescence, enters the cell but is served at similar levels in mitochondria expressing all geno- restricted from the mitochondria unless the mPTP is open. types of APOL1, demonstrating that general mitochondrial In the absence of CoCl2,calceinfluorescence was similar import of proteins remained intact (Supplemental Figure 7). in cells expressing either G0 or RV APOL1. (Figure 5, A and B). However, after addition of CoCl2,calceinfluores- APOL1 G0 and RVs Bind mPTP Components, but Only cence was reduced in G1- and G2-expressing cells but not RVs Activate Pore Opening in G0-expressing cells (Figure 5, C and D), indicating in- To explore how mitochondrial APOL1 alters mitochondrial creased mitochondrial permeability to CoCl2 and suggesting function and drives cell death, we performed immunoprecip- increased mPTP opening in G1 and G2 cells only. As mPTP ac- itation mass spectrometry to identify potential mitochondrial tivation should also lead to rapid mitochondrial depolarization,

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Table 1. APOL1 immunoprecipitate contains multiple mPTP- activate pore opening, leading to cell death. These data directly associated proteins address several important, unanswered questions related to Total Peptides APOL1 biology. Protein G0 G1 G2 First, it has been surprising that investigators have not identified clear, consensus differences between APOL1 G0 and RVs ATP5B 56 72 40 ATP5A1 51 51 62 subcellular localization using a range of microscopy tech- ATP5C1 9 16 12 niques. Similarly, although candidate proteins have been pro- ATP5F1 5 3 1 posed, investigatorshave not identifiedAPOL1protein binding ATP5O 3 5 7 partners that consistently bind only (or with major affinity SLC25A3 28 20 27 differences) to G0 versus RVs across studies.28,48 We show SLC25A4 17 25 29 here that both non-risk and RV APOL1 appear to follow sim- SLC25A5 14 25 17 ilar mitochondrial import pathways. However, once inside the SLC25A6 5 6 5 mitochondria, behavior diverges. G0 remains mostly mono- APOL1 was immunoprecipitated from HEK293 cells stably expressing G0, meric whereas RVs appear to form a range of higher-order G1, or G2 and mass spectrometry was performed to identify potential APOL1 binding partners. Identified candidate binding partners were highly enriched oligomers. Blocking mitochondrial APOL1 import and aggre- for mitochondrial proteins. Total peptide numbers are shown for the MPTP- gation also prevents APOL1-induced cell death. These find- associated proteins among them. The criteria for assignment of mitochon- ings may explain how the lack of differences in subcellular drial proteins is described in the Methods and the full list of mitochondrial fi proteins identified by mass spectrometry is listed in Supplemental Table 2. localization of G0 versus RVs and the lack of identi ed proteins uniquely binding G0 or RVs can be compatible with we measured mitochondrial membrane potential using marked differences in cytotoxicity of G0 and RVs expression: 46 both G0 and RVs are imported into the matrix and both as- TMRM dye. Consistent with the CoCl2 quench data, G1 and G2 cells showed reduced TMRM staining, whereas fluo- sociate with mPTP machinery in the IMM, with different rescence remained strong in EV and G0 cells, indicating in- consequences. tact mitochondrial membrane potential in EV and G0 cells Second, the differing propensities of G0 versus RVAPOL1 to (Figure 6A). oligomerize point toward potential mechanisms that may We tested the effect on APOL1-associated cytotoxicity of eventually illuminate recessive gain-of-function toxicity. Al- CsA, an mPTP inhibitor that binds CYPD and prevents though much additional experimentation will be required to mPTP opening.47 We observed that CsA rescued G1- and understand APOL1-APOL1 interactions in their full complex- G2- mediated cytotoxicity in a dose-dependent manner ity, the ability of G0 to alter RVs self-interaction may be central (Figure 6B) without affecting APOL1 protein levels (Supple- to why RV toxicity from one allele could be inhibited by G0 mental Figure 9A). To ensure CsA rescue was attributable to expression from the other allele, and why humans heterozy- CYPD binding and not to calcineurin inhibition, we treated gous for APOL1 RVs do not develop rates of disease interme- cells with NIM811, a CsA analog that potently inhibits CYPD diate between G0 and RV homozygotes. Our native PAGE gels without binding calcineurin. NIM811 also rescued G1 and G2 reveal APOL1-containing oligomeric complexes of varying mediated cytotoxicity in a dose-dependent manner (Figure sizes (up to about 1.2 MDa) as well as aggregates too large to 6C) without effect on APOL1 protein levels (Supplemental escape from the stacking gel. Itwill be important in the future to Figure 9B). We then tested cytotoxicity after knockdown of directly demonstrate that APOL1 oligomerization is necessary fi several mPTP components or modulators (Figure 6D) that and suf cient to cause cell death, and to isolate the toxic APOL1 were identified by APOL1 by immunoprecipitation mass spec- species. Neurologic diseases caused by aggregate formation trometry assay (and that the cell was most likely to tolerate). present several models to consider. For example, the theory fi Knockdown of SLC25A3, A4, and A5 all markedly reduced that large molecules consisting of amyloid brils cause disease fi APOL1 RV–induced cell death. Similarly, knockdown of is giving way to the idea that toxic pre brillar oligomers may be CYPD (the target of CsA and a well established activator of the pathogenic entity in Alzheimer, Parkinson, and amyotro- 49,50 fi the mPTP) also rescued APOL1 RV-expressing cells from phic lateral sclerosis disease. These pre brillar oligomers death. These data indicate that both G0 and RV APOL1 can can form pores and drive neuron death, suggesting avenues to interact with the mPTP, but mPTP is preferentially activated explore regarding the effect of APOL1 on podocytes, a cell type by RV APOL1. with many similarities to neurons. Third, our data appear to have important implications for the perplexing effects of APOL1 on mitochondrial respiration. DISCUSSION We have replicated the interesting observation that G1 and G2 impairs mitochondrial respiration, whereas G0 may in some Our data demonstrate a pathway that begins with mitochon- cases enhance it, and we at least partially reversed these effects drial APOL1 import and leads to binding of an IMM complex by blocking new APOL1 mitochondrial import.27,32 In a non- related to the mPTP (Figure 7). APOL1 mitochondrial import hypothesis-based, mass spectrometry experiment we saw alters mitochondrial metabolism, and APOL1 RVs eventually that APOL1 of all genotypes pull down the fundamental

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A Calcein Mitotracker Merge C Calcein Mitotracker Merge

EV EV

G0 G0

G1 G1

G2 G2

B 1×107 n = 173-216 D 1×106 n = 89-106 8×106 8×105 6×106 6×105 4×106 4×105 Calcein Calcein 2×106 2×105 fluorescence (AU) fluorescence (AU) 0 0 EV G0 G1 G2 EV G0 G1 G2 unquenched +CoCl2

Figure 5. APOL1 RVs induce MPTP opening (A). Calcein staining before CoCl2 quench was not different between EV, G0, G1 and G2 cells. (B) Quantification of calcein fluorescence before addition of cobalt chloride. Data expressed as mean6SD, for “n” cells subjected to quantification. (C) Calcein-AM staining with CoCl2 quenching indicates that APOL1 RVs cause opening of the mPTP. Calcein-AM dye diffuses into and throughout the cell and is activated upon esterase cleavage of the acetoxymethyl ester group in live cells (green staining). CoCl2 enters the cell but, under normal conditions, cannot enter the mitochondria. mPTP activation leads to pore formation and allows cobalt chloride to enter the mitochondria. Confocal imaging demonstrates decreased calcein fluorescence in G1- and

G2-expressing cells after CoCl2 quenching, indicating MPTP pore opening. Images shown are maximum intensity projections of a representative z-stack. (D) Quantification of mPTP pore induction as a function of APOL1 genotype, as measured by calcein fluorescence after CoCl2 as described in (B) above. Data expressed as mean6SD, for “n” cells subjected to quantification. Original magni- fication, X630.

ATP-generating complex of the cell, the mitochondrial ATP mPTP, an event that precedes neuronal cell death. Parallel synthase, a ﬁnding we verify with immunoprecipitation mechanisms involving self-aggregation in two types of com- Westernblotsperformedinthereversedirection.Direct plex, terminally differentiated cells with limited capacity for interaction of both G0 and RVs with the ATP synthase, but repair or regeneration suggest that terminally differentiated in different oligomeric states, provides a potential basis for cells may lack intrinsic resistance to this type of injury. opposing changes in mitochondrial respiration. A remark- Finally, the convergence of APOL1 at the mPTP provides a able precedent is the recent demonstration that a-synuclein candidate early mechanismfor triggeringcell deaththat may, in also binds to the ATP synthase.51 Whereas monomeric part, explain the wide range of downstream cell death mech- a-synuclein binding to ATP synthase improves the efﬁciency anisms that have been attributed to APOL1. Although the exact of ATP synthesis, binding of a-synuclein oligomers impairs nature and subunit composition of the mPTP have been sub- ATP generation and ultimately leads to activation of the jects of contention for decades, evidence strongly supports a

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A EV G0 G1 G2 B 1.2 DMSO 0.1uM CsA 1.0 1uM CsA TMRM 0.8

0.6

0.4 Cytotoxicity/Viability

0.2 TMRM +DAPI

0.0 C D EV G0 G1 G2 1.0 DMSO 0.20 EV 0.5 G0 0.1uM NIM811 1uM NIM811 0.4 0.15 0.8 0.3 0.10 0.6 0.2

0.05 Cytotoxicity/Viability Cytotoxicity/Viability 0.1 0.4 0.00 0.0 Cytotoxicity/Viability

0.2 siNT siNT siCYPD siApol1 siCYPD siApol1 siSLC25A3siSLC25A4siSLC25A5 siSLC25A3siSLC25A4siSLC25A5

0.0 1.2 G1 1.4 G2

EV G0 G1 G2 1.0 1.2 1.0 0.8 ** 0.8 0.6 ** ** 0.6 0.4 ** 0.4 ** ** ** ** Cytotoxicity/Viability Cytotoxicity/Viability 0.2 0.2 ** ** 0.0 0.0

siNT siNT siCYPD siApol1 siCYPD siApol1 siSLC25A3siSLC25A4siSLC25A5 siSLC25A3siSLC25A4siSLC25A5

Figure 6. APOL1 RVs depolarize mitochondrial potential and induce cytotoxicity, reversible with pharmacologic inhibition or knockdown of mPTP components. (A) TMRM dye accumulates in the mitochondrial matrix on the basis of the hyperpolarized mitochondrial transmembrane potential. Compared with EV and G0 cells, TMRM ﬂuorescence is much lower in APOL1 RV-expressing cells, indicating reduced mitochondrial transmembrane potential in RV cells. Original magniﬁcation, X630. (B) CYPD inhibitor CsA blocks mPTP formation and reduces APOL1 RV–induced cell death. Each dot represents an independent experiment. For both G1 and G2 cell lines, DMSO versus 0.1 mM CsA and DMSO versus 1 mMCsA;P,0.001. (C) NIM811, a CsA analog that can inhibit MPTP by binding to CYPD but cannot bind to calcineurin, blocks APOL1 RV induced cell death. Each dot represents an independent experiment. For both G1 and G2 cell lines, DMSO versus 0.1 mM NIM811 and DMSO versus 1 mMNIM811;P,0.001. (D) Knockdown of individual MPTP pore components or modulators blocks APOL1-induced cell death. Supplemental Figure 8 includes immunoprecipitation Western blots demonstrating interaction between APOL1 and MPTP components. Each dot represents an independent experiment. **P,0.001 comparing each siRNA to siNT-treated cells. model in which the ATP synthase, SLC25A3, SLC25A4, and observe direct binding of APOL1 to these mPTP complex SLC25A5 all have important roles, either as components of components, but we found that individual knockdown of sev- the pore itself or as key modulators.43–45 Not only did we eral (for which knockdown was expected to be compatible

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among the earliest detectable cellular disturbances temporally after APOL1 RV expression.32 The very early occurrence of mitochondrial dysfunction, our demonstration that cell death depends on mitochondrial APOL1 import and mPTP activation, and the marked differences in APOL1 variant aggregation that appear to occur inside mitochondria together ATP Synthase support the idea that mitochondrial injury could be the primary cytotoxic event triggered by the APOL1 RVs. After ? Misfolding; mitochondrial APOL1 RVs initiate global cellular events such aggregation mPTP as calcium release through the mPTP, many processes may regular Opening become dysregulated and downstream cell death effector re-folding; mechanisms activated (e.g.,ionfluxes, kinases, autophagy, monomers APOL1 and apoptosis, just to name a few). Because APOL1-mediated import disturbances in other organelles could also, in theory, cause ATP-depletion and altered mitochondrial function as a sec- HSPA9 ondary event, the exact relationships between APOL1- induced mitochondrial injury and alteration of membrane 17 23 currents, kinase activation, and regulation of processes such as autophagy remain to be fully worked out. The different clin- 22 ical phenotypes observed in various APOL1 nephropathies— rapid versus indolent, proteinuric versus nonproteinuric, with a variety of histologic patterns of injury in the glomerulus— 40 22 70 leave open the possibility of more than one mechanism in 20 more than one target cell type. APOL1 G0 APOL1 RV The experiments here present a basic molecular model for Figure 7. Model for differential toxicity of G0 and RV APOL1. APOL1 kidney disease but leavenumerous follow-up questions APOL1 G0 and RVs enter the mitochondrial matrix via the same that need to be answered. The critical APOL1 mitochondrial import machinery. Although they remain mostly monomeric, RVs localization signal sequence is not yet known and identifying tend to form higher-order oligomers after import. Whereas all it will be important evidence to support any mitochondria- APOL1 variants appear to bind components or regulators of centric APOL1 injury model. Identifying the proteins or lipids the mPTP, the binding of aggregated RVs (likely in oligomeric that ferry APOL1 to the mitochondria, and reversing the cell form) activates pore opening, mitochondrial dysfunction, and cell death phenotype by blocking their transport function, will also death. Because proteins generally unfold before mitochondrial help support mitochondrial initiation of cellular injury. A import, one possibility is that G0 refolds normally after import deeper understanding of the nature of the APOL1-APOL1 and remains monomeric whereas RVs refold abnormally and have interactions is a high priority, including defining the relevant apropensitytoaggregate. binding domains, the relativeaffinitiesbetween APOL1 protein of different genotypes, and other proteins that may be part of with cell survival) blocked APOL1-induced cell death. Lend- the higher-order APOL1 complexes. Determining the precise ing additional support, we found that knockdown of the ma- molecular events governing the interaction between APOL1 trix protein CYPD, a well validated mPTP regulator, also (both monomeric and oligomeric) and the ATP synthase will blocked cell death, as did the CYPD inhibitor CsA, a drug be required to establish with certainty that oligomeric RV- known to be useful clinically in a subset of patients with containing complexes directly cause mPTP activation and 52 FSGS. Our CoCl2 calcein fluorescence quench experiments cell death. Other APOL1 mitochondrial binding partners iden- provide functional evidence that the mPTP is activated in tified in our mass spectrometry experiments may also be sub- the setting of RV APOL1 expression. We find it intriguing stantive factors in causing mitochondrial dysfunction, and that aggregated proteins have been proposed to play a role in excluding them or defining their contribution is needed mPTP function long before the demonstration of a-synuclein for a more complete understanding of APOL1 behavior in multimers binding to ATP synthase.53 Because mPTP opening mitochondria. leads to Ca2+ efflux from mitochondria into cytosol, it may We will also ultimately have to demonstrate that similar trigger a range of downstream cell death effector pathways and processesareoccurringinhumanswithAPOL1-mediatedkidney provide a unifying candidate mechanism driving the several disease and answer a range of related questions: why do APOL1 modes of APOL1-associated cell death observed in previous RVs only seem to have deleterious effects on kidney cells? Does reports. low APOL1 glomerular expression in kidney biopsies from some Our data add strong mechanistic support at the molecular patients with APOL1-related diseases reflect podocyte death due level to the key observation that mitochondrial dysfunction is to high APOL1 expression (gain-of-function) or does it suggest

2366 JASN JASN 30: 2355–2368, 2019 www.jasn.org BASIC RESEARCH that there may be loss-of-function effects of RVs as well? Does Supplemental Figure 4. siRNA Validation blots for knockdown APOL1 cause kidney disease in podocytes or other kidney cell efﬁciencies not shown in main ﬁgures. types by suppressing mitochondrial function, by activating pore Supplemental Figure 5. APOL1 can bind to other APOL1 openingwhenitbindstothemPTP,orbyotherprocesses?Whatis molecules. the second hit that initiates the APOL1 kidney disease process in Supplemental Figure 6. TOMM20 knockdown reduces APOL1 individuals with two RV? Additional experiments in other cell oligomerization, whereas TOMM70 and TIMM22 knockdown does systems, animal models, and human tissue represent important not alter APOL1 oligomerization. next steps in understanding the effects of APOL1 on mitochon- Supplemental Figure 7. G1 and G2 do no inhibit import of newly drial function and its importance in the different phenotypes synthesized protein into the mitochondria. driven by APOL1 RVs. Supplemental Figure 8. APOL1 interacts with multiple MPTP components. Supplemental Figure 9. CsA and NIM811 treatment does not affect APOL1 protein levels. ACKNOWLEDGMENTS

Shah and Friedman conceived the project, designed the experiments, analyzed results, and wrote the manuscript. Shah, Lannon, Dias, and REFERENCES Zhang performed the experiments. Dias, Lannon, Alper, and Pollak proposed experiments, provided scientiﬁc insight and analysis, of- 1. Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, Freedman BI, et al.: Association of trypanolytic ApoL1 variants with kidney disease fered critical review, and edited the manuscript. in African Americans. Science 329: 841–845, 2010 We would like to thank Lay-Hong Ang at the Beth Israel Deaconess 2. Tzur S, Rosset S, Shemer R, Yudkovsky G, Selig S, Tarekegn A, et al.: Medical Center (BIDMC) Imaging Core, Xiaowen Liu at the BIDMC Missense mutations in the APOL1 gene are highly associated with end Metabolism and Mitochondrial Research Core, and Ross Tomaino stage kidney disease risk previously attributed to the MYH9 gene. Hum – at the Harvard Medical School Taplin Mass Spectrometry Facility Genet 128: 345 350, 2010 3. Kopp JB, Nelson GW, Sampath K, Johnson RC, Genovese G, An P, et al.: for guidance with our experiments. APOL1 genetic variants in focal segmental glomerulosclerosis and HIV- associated nephropathy. J Am Soc Nephrol 22: 2129–2137, 2011 4. Friedman DJ, Kozlitina J, Genovese G, Jog P, Pollak MR: Population- DISCLOSURES based risk assessment of APOL1 on renal disease. J Am Soc Nephrol 22: 2098–2105, 2011 5. Duchateau PN, Pullinger CR, Cho MH, Eng C, Kane JP: Apolipoprotein Dr. Friedman and Dr. Pollak are co-inventors of patents related to APOL1 L gene family: Tissue-speciﬁc expression, splicing, promoter regions; diagnostics and therapeutics, own equity in Apolo1Bio, and have research discovery of a new gene. J Lipid Res 42: 620–630, 2001 funding from and consulted for Vertex. 6. Smith EE, Malik HS: The apolipoprotein L family of programmed cell death and immunity genes rapidly evolved in primates at discrete sites of host-pathogen interactions. Genome Res 19: 850–858, 2009 FUNDING 7. Molina-Portela Mdel P, Lugli EB, Recio-Pinto E, Raper J: Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes. Mol Biochem Parasitol 144: 218–226, 2005 This work was supported by funds from the US Department of Defense 8. Pérez-Morga D, Vanhollebeke B, Paturiaux-Hanocq F, Nolan DP, Lins L, (W81XWH-14-1-0333), National Institutes of Health (MD007898), the Homblé F, et al.: Apolipoprotein L-I promotes trypanosome lysis by NephCure Foundation, Vertex Pharmaceuticals, and the Ellison Foundation. forming pores in lysosomal membranes. Science 309: 469–472, 2005 9. Xong HV, Vanhamme L, Chamekh M, Chimfwembe CE, Van Den Abbeele J, Pays A, et al.: A VSG expression site-associated gene con- SUPPLEMENTAL MATERIAL fers resistance to human serum in Trypanosoma rhodesiense. Cell 95: 839–846, 1998 10. Uzureau P, Uzureau S, Lecordier L, Fontaine F, Tebabi P, Homblé F, This article contains the following supplemental material online at et al.: Mechanism of Trypanosoma brucei gambiense resistance to http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019020114/-/ human serum. Nature 501: 430–434, 2013 DCSupplemental. 11. Friedman DJ, Pollak MR: Genetics of kidney failure and the evolving Supplemental Table 1. APOL1 Sequences used to make Tet-in- story of APOL1. J Clin Invest 121: 3367–3374, 2011 ducible HEK293 cells. 12. Thomson R, Genovese G, Canon C, Kovacsics D, Higgins MK, Carrington M, et al.: Evolution of the primate trypanolytic factor Supplemental Table 2. List of mitochondrial proteins pulled down APOL1. Proc Natl Acad Sci U S A 111: E2130–E2139, 2014 after APOL1 immunoprecipitation mass spectrometry. 13. Cooper A, Ilboudo H, Alibu VP, Ravel S, Enyaru J, Weir W, et al.: APOL1 Supplemental Figure 1. Western blot analysis of organelle markers renal risk variants have contrasting resistance and susceptibility asso- in the cytosolic and mitochondrial-enriched fractions. ciations with African trypanosomiasis. Elife 6: e25461, 2017 Supplemental Figure 2. APOL1 translocation to mitochondrial 14. Friedman DJ, Pollak MR: Apolipoprotein L1 and kidney disease in Af- rican Americans. Trends Endocrinol Metab 27: 204–215, 2016 matrix is independent of carrier pathway OMM protein TOMM70. 15. Johnstone DB, Shegokar V, Nihalani D, Rathore YS, Mallik L, Ashish, Supplemental Figure 3. TOMM20 knockdown can partially rescue et al.: APOL1 null alleles from a rural village in India do not correlate APOL1 RV–mediated mitochondrial dysfunction. with glomerulosclerosis. PLoS One 7: e51546, 2012

JASN 30: 2355–2368, 2019 Variant APOL1 Activates Mitochondrial Pore Opening 2367 BASIC RESEARCH www.jasn.org

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