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. 2020 Aug 20;23(9):101476. doi: 10.1016/j.isci.2020.101476

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© 2020 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Role of TbKIFC1 in Membrane Trafficking

(A) WT and VHS7 mutant sequences of the TbKIFC1 VHS domain, with indication of the different helices (H1–H8). The asterisks point to the mutations in VHS7.

(B) Model of the VHS domain, built by homology modeling with 1ELK and 1X5B (PDB codes) as templates. The inset shows the H7-H8 helical hairpin (TbKIFC1 H7H8). Lower panel: model of helices H7H8 highlighting residues V103, I107, V111, W114, and Y118, mutagenized into A in VHS7.

(C) Immunodetection of recombinant VHS (0.5 μg/mL) association with various lipids spotted on membrane strips (LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PI(3)P, PI(3)phosphate; PI(4)P, PI(4)phosphate; PI(5)P, PI(5)phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PI(3,4)P2, PI(3,4)bisphosphate; PI(3,5)P2, PI(3,5)bisphosphate; PI(4,5)P2, PI(4,5)bisphosphate; P(3,4,5)P3, PI(3,4,5)trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine; TG, triglyceride; DAG, diacylglycerol; PG, phosphatidylglycerol; CL, cardiolipin; Chol, cholesterol; SM, sphingomyelin; Blank, no lipid).

(D) Adsorption of recombinant VHS domain into an air-water interface (without lipids) or into a lipid monolayer with DMPC, DMPC-DMPS (1:1), or DMPC-DMPS-cholesterol (1:1:2). Top panel: kinetics; bottom panel: variation of surface pressure at equilibrium. Data are represented as mean ± SD; n = 3.

(E) Molecular dynamics of the interaction between the TbKIFC1 H7H8 peptide and a membrane composed of PC/PS (9:1). In the top section of the figure, the peptide is represented as in (B) (lower panel), and the membrane is represented by a line joining the phosphate groups. The bottom section shows the radial distribution function of POPC (black line) and POPS (red line) around the peptide mass center (MC).

(F) Interaction with PS. The adsorption of the H7H8 helices of TbKIFC1 into a lipid monolayer composed of DMPC with or without DMPS (1:1) was evaluated by measuring maximal insertion pressure (MIP, dark gray bar) and attractiveness factor (ΔΠ, light gray bar). The WT and VHS7 mutant version of H7H8 (see A) are compared. MIP corresponds to the x-intercept of the linear regression (inset) of ΔΠ_eq (surface pressure at the equilibrium) versus Π_i (initial pressure). Data are represented as mean ± SD; n = 3.

(G) Relative expression levels of the endogenous or ectopic TbKIFC1 genes in various TbKIFC1^RNAi cell lines with or without addback TbKIFC1expression (WT or VHS7), as measured by quantitative RT-PCR. Data are represented as mean ± SEM; n = 3. (addb refers to the different recoded TbKIFC1 addback sequences reinserted in the RNAi line, provided or not with a biotin ligase [BL] tag).

(H) Involvement of H7 in APOL1 transport, as measured by trypanolysis as a function of rAPOL1 concentration during incubation with control (CTRL) or TbKIFC1^RNAi parasites. Data are represented as mean ± SEM; n = 3.