A Cell Biologist's View on APOL1: What We Know and What We Still Need to Address

Abstract

APOL1 is the most recent member of the APOL gene family and is expressed exclusively in humans and a few higher primates. More than twenty years ago, it was discovered that APOL1 protects humans from infections by trypanosome subspecies that cause African sleeping sickness. Interestingly, by a co-evolutionary process between parasite and host, two APOL1 variants emerged, which, in addition to their trypanotoxic effects, are simultaneously associated with a significantly increased risk for various different kidney diseases, which are now summarized as APOL1-mediated kidney diseases (AMKDs). The aim of this review is to highlight and formulate key aspects of APOL1's cell biologic features, including questions and unaddressed aspects. This perspective may contribute to a deeper understanding of APOL1-associated cytotoxicity as well as AMKDs.

Keywords: AMKD; APOL1; APOL1-mediated kidney disease; Apolipoprotein L1; ion pore; nephrotoxicity; renal risk variants; topology.

Figure 1

Structural features of APOL1. (a) The regions described first relate to functional domains of APOL1. Besides the signal peptide (SP) in the N-terminus, important for secretion, the sequence contains a pore forming domain (PFD), which includes the Bcl-2 homology 3 (BH3) domain-like sequence motif (BSM) associated with putative anti-, or pro-apoptotic features. The PFD and neighboring membrane-addressing domain (MAD) enable the membrane localization and pore activity. C-terminally localized is the SRA-interacting domain (SID). SID is important for SRA binding. Naturally occurring variants of the African haplotype (K150E, M228I, R255K) are located in the PFD and MAD, while renal risk variant G1 (S342G, I384M) and G2 (ΔN388,389) are localized in the SID. In turn, modifier 1 (M1; N264K) which prevents G2-mediated toxicity is localized in the MAD. (b) A more topological classification of APOL1 regions focusses on up to four putative transmembrane helices (TM1-4) in addition regions to the SP, which determines the topology of the APOL1 splice variants. Juxtaposed to the BSM, TM1 and TM2 depict the membrane insertion domain (MID) followed by TM3 harboring the N264K variant and TM4 also defined as the pore lining region (PLR) enabling pH-sensitivity and selectivity of the pore. Here, one of the G1 variants (S342G) is located, while the other (I384M) appears together with the deletions causative for G2 in a leucine zipper (ZIP) domain in the C-terminus, probably involved in pore formation via APOL1 multimerization. (c) A mixed functional-topological model by Pays and colleagues includes a combination of a hydrophobic cluster (HC) paired with a close-by ZIP domain located in the N-terminal part as well as in the C-term. APOL1 also contains two putative cholesterol recognition amino acid consensus (CRAC) sites in the MAD and the HC2. Following this model, membrane insertion is enabled by TM1, TM2 and the MAD.

Figure 2

Topologies of APOL1 at membranes. The colors used in this figure for characteristic APOL1 features (domains, variants, etc.) corresponds to that of Figure 1. (a) In the 4-TM concept, four transmembrane regions or helices (TMR1-4) span the ER membrane. In the two and a half-spanning loop model (2 ½ TM) two TMRs are present in the PFD, followed by a region between MAD and SID that “dips” into the membrane. Secreted or luminal pools of APOL1 contain a signal peptide (which is cleaved inside the ER, cs) and are only associated (but not inserted) into intracellular membranes. In these three models, all sequence motifs associated with APOL1-linked cytotoxicity (including the BSM, the RRVs, G1 and G2, the amino acids that determining the African haplotype, as well as the two cholesterol-binding sites; see Figure 1) facing the luminal side of intracellular membranes. The fourth concept refers to a cytoplasmic-faced APOL1 topology with only two TMRs (or membrane associated regions), resulting in an orientation in which both the N- and C-terminus as well as all mentioned cytotoxicity-associated sequence motifs facing the cytoplasm (like APOL2). (b) In case that pore formation requires dimerization or even multimerization both the 4 TM and 2 ½ TM concepts facilitate a “tail-to-tail” multimerization via a C-terminal ZIP domain.

Figure 3

Localization, trafficking and secretion of APOL1. The main pools of APOL1 are detected at the endoplasmic reticulum (ER) with further portions reported at the plasma membrane (PM), early/late endosome (EE/LE, also called multivesicular body, MVB), lysosome and endolysosome (EL), mitochondria and Golgi apparatus as well as at Lipid droplets (LD) and high-density lipoprotein (HDL) particles. Recycling and degradation could be mediated via the endolysosomal-autophagic system. Acidification of APOL1 may take place in the endolysosomal pathway or the conventional secretion via the Golgi apparatus (turquoise arrows). Mitochondrial APOL1 could integrate both into the outer or inner mitochondrial membrane (OMM, IMM) and transported via mitochondria-derived vesicles (MDV). Possible pathways for APOL1 secretion (orange arrows) could be conventional secretion (a), or unconventional secretion (b). In addition, transport via LD or HDL particles is possible (thick black arrows). Both arise within the ER, which leads to a lipid mono- or bilayer perhaps containing ER-located APOL1 proteins encapsulating them when released (c). A reported association of APOL1 with cholesterol, which is produced in the ER may also be part of the trafficking and release of APOL1 (d). Inset: Independent of its localization, APOL1 can appear as a membrane-associated protein (in luminal or cytoplasmic orientations), as secretory or luminal protein, as an integral transmembrane protein, or even as pore-forming protein.

Figure 4

Membrane contact sites between organelles as potential APOL1 transfer hubs. Membrane connections between different organelles in close proximity are mediated via membrane contact sites (MCSs) and influence signaling, ion homeostasis, metabolism, stress responses as well as function, division and regulation of organelles. Such MCSs have been identified between ER and lysosomes (a), endosomes (b), mitochondria (c), peroxisomes (d), the Golgi network (e) or lipid droplets (f). MCSs can also be formed between the PM and ER (g) as well as PM and mitochondria (h). APOL1 localizes in/at nearly all the compartments mentioned and could either play a direct role in these MCSs or be transferred there from the cytoplasm or one compartment into another. Such a way of transfer has been described e.g., for mitochondrial protein import. Interestingly, APOL1 was already reported to play a role in mitochondrial fission and mitophagy.

Figure 5

The function of APOL1 as an ion pore. APOL1 pathomechanism includes ion pore functions of the APOL1 renal risk variants. The first possibility could be that the TM-helices of APOL1 themselves form a pore within the PM (a). Pores could also be built by homodimerization of APOL1 subunits (b), or by heterodimers composed of APOL1 and a second protein (c). As a APOL1clustering at the surface has been reported, numerous APOL1 proteins could also be subunits in a multimerized assembly into a megapore (d). Some studies suggest a pH-dependent formation of the pore, which would require involvement of the endolysosomal pathway to acidify and open the APOL1 pore (e). Several experiments hint towards a pore selectivity for cations which could for example facilitate Ca²⁺ flux or K⁺ efflux combined with Na⁺ influx (f). Indirect effects due to activation of nearby ion channels could also be possible (g). All mechanisms bear the possibility that APOL1 not only serves as pore at the PM, but also at other membrane-containing compartments leading to lysosomal or mitochondrial dysfunction or ER stress.