Fig 1: APOL1 C-Terminal Variation Influences APOL3 Binding to APOL1 and NCS-1(A) BLI measurements of interaction between bound APOL3 and recombinant WT APOL1 or APOL1 variants, either in SID1 (LZ1mut) or SID2 (APOL1?, G1, G2) (n = 3).(B) Interactions between APOLs and APOL3 (top panel) or NCS-1 (bottom panel), as measured in the E. coli pDUET co-expression system. Nickel binding of S-tagged proteins reflects their association with His-tagged partners (highlighted within red frames). A1, ?, and ?1 = APOL1 60-398, 60-353, and 60-342, respectively; A3 = APOL3; 3N = APOL3+1% NP-40 instead of 1% CHAPS; M3 = APOL3 HC2-like mutant HCmut. These data are representative of at least 5 independent experiments.(C) Western blot analysis of APOL1, TriFLAG-tagged APOL3, and NCS-1 immunoprecipitates from podocytes. All of the extracts were treated with 1% CHAPS.(D) BLI measurements of interactions between bound NCS-1 and APOL3 under various calcium concentrations (n = 3).(E) BLI measurements of interactions between bound NCS-1 and various recombinant APOLs, either WT or variant in SID1 (LZ1mut) or in SID2 (APOL1?, G1, G2), performed in the presence of 50 µM CaCl2 (n = 3).(F) BLI measurements of interactions between bound NCS-1 and APOL3, in the presence or absence of either 1% NP-40 or 1% CHAPS (n = 3).(G) BLI measurements of interactions between bound NCS-1 and APOL3 or PI4KB, added sequentially as indicated (n = 3).(H) BLI measurements of interactions between bound NCS-1 and APOL3, either alone or mixed with different molar ratios of APOL1 (left panel) or APOL1? (right panel) (n = 3).
Fig 2: APOL3 Exhibits K+ Channel Activity Influencing Intracellular Ca2+ Levels(A) APOL3 channel activity in planar soy lipid membranes (n = 5).(B) Model of APOL-mediated ion currents in the podocyte ER and Golgi membranes.(C) Effects of APOL1 or APOL1? on APOL3 channel activity. APOL3, APOL1, and NCS-1 were added at 56, 59, and 109 pmol/mL (n values indicated in the table).(D) Quantification of the peak of cytosolic Ca2+ increases in response to different ATP concentrations (n = 5). The values were normalized to the signal obtained using 100 µM ATP.(E) Intracellular Ca2+ store contents. In the top panels, each curve is representative of one single experiment (calculated from three technical replicates). In the lower panels, data were quantified by calculating the area under the curve (AUC) normalized versus the WT cell line (black line). In the dot plots, each cell line is represented with a different color and each independent experiment is represented with a different symbol (left panel, n = 9; center and right panels, n = 7). Data belonging to the same experiment (e.g., 96-well plate) are indicated with the same symbol.
Fig 3: SID1 Interactions with SID2 Inhibit HC Exposure in APOL1(A) Sequences of the synthetic peptides used for SPR measurements (SID1p and LZ1p = APOL1 K73-K125 and L88-K125; SID2p, G1Cp, G2Cp = APOL1 G332-L392 from WT or variant sequences; A3Cp = APOL3 G265-L325). Violet and pink colors highlight residues defining the hydrophobic clusters (HC, violet) and leucine zippers (LZ, pink). Yellow and green colors, respectively, indicate the N-term tag used for coupling the peptides to the SPR chip and residues specific to G1 and G2 variants. In A3Cp, residues evoking the APOL1 G1 or G2 variants are shown in blue.(B) SPR-determined binding parameters for the interactions of the SID1p and LZ1p peptides to various immobilized peptides.(C) SPR sensorgrams of interactions between SID peptides.(D) Electrophoretic mobility shift of APOL1?. A1, ? and ?1 = APOL1 60–398, 60–353, and 60–342, respectively (expected sizes: 41.3, 34.4, and 32.2 kDa). The sequence of the APOL1? band excised from the SDS-PAGE gel was determined by mass spectrometry to check its integrity (peptide coverage in yellow; oxidized methionines in green). The HC2 sequence is underlined, and the C terminus of APOL1?1 is denoted by an arrow.(E) Nile red staining of recombinant APOL1, G1, or G2 (1 µM each). Fluorescence emitted by Nile red (0.01 µM) was measured at 630 nm (fluorescence of APOL1 was set to 1). Error bars, SDs; n = 9.(F) Model for the interactions between SIDs in APOL1. LZ2 with a star, mutant leucine zipper, exhibiting reduced interaction affinity with LZ1. K+ refers to channel activity (parentheses denote inactivation).(G) Model for the interactions between APOL3 and NCS-1 (top) or APOL1?+NCS-1 (bottom). The APOL3 sequences homologous to APOL1 HCs and LZs are indicated in italics. K+ refers to channel activity (parentheses denote inactivation).
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