Fig 2: APOL1 vA gene structure and regions with positively selected sites. (A) Amino acids (single letter code) and coordinates mark positively selected sites for APOL1–4 derived from at least two of three PAML models (M2/M8/BS) as large stars. The positively selected sites found by Smith and Malik (2009) are indicated for APOL1–2, APOL3–4, and APOL6. Red labeled letters show overlapping sites of different studies. The APOL1 variation of alternative amino acids or deletion sites (?) are shown above the exon structure (gray). APOL1 was previously divided into four functional regions, an SP, a PFD, an MAD, and the SID (green boxes). The PFD also contains a putative pro-apoptotic BH3-only domain sequence motif (BSM). More recent conceptions suggest an SP (gray bar) and up to four TMs (SP, gray and TM1-4, black bars with their coordinates indicated as numbers). The first two TMs are also referred to as membrane insertion domain (MID) or helix–turn–helix region (H-L-H). The TM4 overlaps with a pore-lining region (PLR), followed by leucine-zipper domain (ZIP). (B) Model of possible orientations of APOL1 (and APOL2) at the ER. The presence or absence of the SP determines the orientation of APOL1 splice variants and of APOL2 at the ER. Functional SPs result in a luminal (cis) ER localization of the APOL1 C-terminus. If APOL1 functions as an ion pore, this most likely requires two (I), “two and a half” (II), or four (III) TMs (black boxes). During evolution, positively selected amino acids accumulate in regions within, or close to, the putative TM3 and TM4 (red arrows). APOL1 vB3 and APOL2, that lack functional SPs (IV), are most likely bound to the cytoplasmic leaflet of the ER membrane (trans). Since cis as well as trans pools of APOL1 show cytotoxic effects—that are pronounced in the case of renal risk variants—our data suggest the presence of different cellular pathomechanisms.

Fig 3: ApoL2 does not regulate cell proliferation or sensitivity to ABT-737. (a) HeLa cells were transfected with control siRNA or siRNA targeting ApoL2 and then they were treated with the BH3 mimetic ABT-737 at 30 µM for 24 h. Cell death was measured by PI incorporation and flow cytometry. Panel shows average and S.E.M. of five independent experiments. (b) HeLa cells were transfected with control 1 siRNA or siRNAs against ApoL2 and growth analysis was performed at indicated time points by crystal violet coloration. The lower panel shows western blot analysis of ApoL2 silencing over time

Fig 4: ApoL2 does not regulate cell death of HeLa cells. (a) Plasmids encoding two different BH3-only proteins (0.5 µg of Bmf and 0.8 µg of Noxa plasmids) were cotransfected with ApoL2 or Bcl-2 and analyzed by microscopy. GFP (0.3 µg) was used as transfection marker. ApoL2 and Bcl-2 plasmids were used at amounts shown. Empty plasmid was used to normalize the amount of transfected DNA. Dead green cells were scored by shrunk morphology and counted from images using fluorescence microscopy. Figure shows average and S.E.M. of three experiments. For statistical analysis, each ApoL2 or Bcl-2 overexpressing condition has been compared with the empty plasmid condition transfected with the same BH3-only protein. NS, nonsignificant. (b, c, d). HeLa cells were transfected with different control siRNAs or siRNA against ApoL2 and then treated with chloroquine, deprived of glucose (glc-), incubated in starvation buffer (EBSS) or treated with tumor necrosis factor (TNF) or actinomycin D (ActD) for 24 h (b, c), or deprived of serum (FBS-) or treated with thapsigargin (Thaps) for 72 h (d). Cell death was measured by PI incorporation by flow cytometry. Figure shows average and S.E.M. of three (d) or five experiments (b, c). Asterisks or NS (nonsignificant) denote significance versus Control 2 (b) or Control 1 (c)

Fig 5: Local APOL gene family and SplitsTree reconstructions for humans and mice. (A) Genomic location of APOL genes from human and mouse with genomic coordinates. Arrows indicate gene orientations. APOLD1 evolved early in vertebrates about 300 Ma, whereas APOL6 was probably inherited by a human–mouse ancestor that lived about 70 Ma. (B) SplitsTree reconstruction of APOL genes based on protein sequences. A similar tree topology was derived by maximum likelihood and Bayesian tree reconstructions (supplementary data SD1, Supplementary Material online). The APOL genes of mice (mm for Mus musculus) are indicated in gray boxes. Black boxes represent human APOL genes (hs for Homo sapiens). The central parallelograms of the reconstruction represent conflicting phylogenetic signals. The tree reconstruction reveals a common origin of mm and hs APOL6 (red boxes). Bootstrap values are shown for representative branches. Balls indicate the clade-supporting integrations of AluJ and AluY elements. The human APOL2 and APOL1 genes share an orthologous AluY element. The orthologous AluY transposons were already present in Catharrine primates (diverged about 30 Ma). The diagnostic AluJ elements were detected at orthologous positions in APOL1-4 in all investigated primates and were probably inherited from a common primate ancestor of about 70 Ma. The double-slash in the mouse locus indicates the exclusion of a large genomic region. Human and mouse APOLD1 are located on chromosomes 12 and 6, respectively, and diverged significantly from other APOL genes.