Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate

Abstract

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

Figure 1

Human hVPS13A expressed in yeast cells does not complement vps13Δ defects. (A) Secretion of CPY. Yeast cultures were spotted on nitrocellulose membrane plated on SC-leu medium, incubated for 14-16 h and washed off. The level of secreted CPY was estimated using anti-CPY antibody. (B) Sensitivity of yeast strains to L-canavanine. Serial dilutions of wild type and vps13Δ strains bearing indicated plasmids were spotted on synthetic minimal medium supplemented with (3 µg ml ^− 1) or without L-canavanine. (C) Degradation of Sna3-GFP protein. Cells as in B were grown in SC-leu-ura medium to log-phase and total protein extracts were analyzed by SDS-PAGE followed by Sna3-GFP and GFP detection with anti-GFP antibody. Ubiquitinated (Ub) Sna3-GFP derivatives are indicated. The ratio GFP/(Sna3-GFP + Sna3-GFP-Ub + GFP) was calculated and is given for each strain as percentage of free GFP. (D) The detection of the hVps13A protein in yeast cells. Proteins extracted from vps13Δ strain expressing VPS13-GFP or hVPS13A-GFP under the TEF1 promoter were immunoprecipitated, subjected to SDS-PAGE and western blot analysis with anti-GFP antibody.

Figure 2

Yeast VPS13 gene bearing the ChAc mutation I2749R does not complement deletion of VPS13 in yeast cells. (A) The homology of Vps13 region containing the ChAc mutation (I2771R in human and I2749R in yeast). Logo diagram showing sequence conservation of the Vps13 orthologs from 15 model organisms was obtained using the WebLogo 3 program (67). Alignment was prepared using Clustal Omega tool (EMBL-EBI). (B) Secretion of CPY. Yeast cultures expressing wild type and mutant VPS13 under control of VPS13 native promoter were grown and analyzed as in Figure 1A. (C) Sensitivity of cells to L-canavanine. Serial dilutions of wild type and vps13Δ strains bearing plasmids containing wild type and mutant VPS13 under the control of VPS13 native promoter were spotted on medium supplemented with (1 µg ml ^− 1) or without L-canavanine. (D) Degradation of Sna3-GFP protein. Cells expressing P_VPS13-VPS13 or P_VPS13-vps13-I2749R were grown in SC-leu-ura medium to log-phase and total protein extracts were analyzed by SDS-PAGE followed by Sna3-GFP detection with anti-GFP antibody. Irrelevant lane was removed. Percentage of free GFP [GFP/(Sna3-GFP + Sna3-GFP-Ub + GFP)] in each lane is given. (E) The level of Vps13-I2749R-GFP in yeast cells. Cells transformed with plasmids bearing P_TEF1-VPS13-GFP or P_TEF1-vps13-I2749R-GFP were grown to log-phase, total protein extracts were prepared and analyzed by SDS-PAGE followed by anti-GFP western blotting.

Figure 3

Vps13 influences the actin cytoskeleton organization and interacts with actin. (A) Organization of actin cytoskeleton in vps13Δ cells. Wild type and vps13Δ cells were grown to log-phase, fixed and stained using labeled phalloidine and observed by fluorescence microscopy. Scale bar, 5 µm. (B). Actin cytoskeleton organization in vps13Δ cells bearing P_VPS13-vps13-I2749R. Assay was performed as in A. (C) Graphic representation of actin cytoskeleton organization in vps13Δ cells. At least 100 cells of each strain were observed and the percentage of cells with well-polarized and non-polarized actin cytoskeleton is indicated. Error bars represents standard deviations for three experiments. (D) Immunoprecipitation of wild type and mutant Vps13-GFP proteins. Yeast cells expressing P_TEF1-VPS13-GFP or P_TEF1-VPS13-I2749R-GFP were disrupted using glass beads and GFP-tagged proteins were precipitated using GFP-Trap magnetic beads. Samples were analyzed by standard SDS-PAGE followed by western blotting using anti-GFP and anti-actin antibodies. The 1/100 volume of extract taken to IP was loaded in Total lanes. Strain bearing empty plasmid or plasmid encoding GFP alone was used as a negative control. Irrelevant lane was removed.

Figure 4

Vps13 fragments bind lipids and I2749R substitution attenuates this interaction. (A) Schematic representation of hVps13A and Vps13 domain structure and fragments used in PIP strip assay. Coomassie stained gel showing purified fragments used for PIP strip assay is also shown. Position of isoleucine (I) residue mutated to arginine (R) in ChAc patient and the corresponding site in yeast protein (I2771 in hVps13A and I2749 in yeast Vps13) is indicated by asterisks. Particular domains are indicated by boxes: black, chorein_N; striped, SHR_BD; grey, APT1; dotted, truncated and full-lengh ATG_C. (B) Binding of Vps13 fragments to lipids. GST-Vps13-(SHR_BD -APT1), GST-Vps13-(APT1), GST-Vps13-(SHR_BD) proteins were expressed in E. coli and purified. Up to 1 µg of purified proteins was used to test for binding to lipids deposited on the membrane (PIP strip). The fusion proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PI3P, PI3-phosphate; PI4P, PI4-phosphate; PI5P, PI5-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, PI4,5-bisphosphate; PI(3,4,5)P₃, PI(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine. Experiment was performed at least two times with proteins from independent purifications. Representative result is shown. (C) Binding of Vps13 fragments to PI- and PI3P-containig liposomes. Crude extracts from E. coli containing wild type (WT) or I2749R mutant (*) variant of GST-Vps13-(SHR_BD -APT1) and GST-Fab1-(FYVE) as a positive control were incubated with indicated biotin-tagged liposomes. Liposomes were pull down using the streptavidin-covered beads and processes for western blot. All pull down fractions and 32 μl of each supernatant were loaded on the gel. The fusion proteins were detected using anti-GST antibody. Experiment was performed three times. Representative result is shown.

Figure 5

The level of PI3P influences the localization of Vps13-GFP. (A) Localization of Vps13-GFP in PI3P deficient vps30Δ and vps38Δ cells. Strains transformed with plasmid bearing P_TEF1-VPS13-GFP were grown in SC-leu minimal medium to log-phase and the localization of Vps13-GFP was observed by fluorescence microscopy. At least 100 cells for each strain were observed. The percentage of cells displaying various Vps13-GFP distribution patterns, illustrated in the right-hand panel, is shown. (B) Colocalization of Vps13-GFP and FM4-64 in vps30Δ strain. The vps30Δ strain was transformed with a plasmid encoding P_TEF1-VPS13-GFP. Cells were grown in SC-leu medium to log-phase and the localization of Vps13-GFP protein was observed by confocal microscopy. FM4-64 was used to visualize the compartments of endocytic pathway. Scale bar, 5 µm. (C) Localization of Vps13-GFP and Snf7-RFP in vps30Δ strain. The SNF7-RFP vps30Δ strain was transformed with a plasmid encoding P_TEF1-VPS13-GFP. Cells were grown in SC-leu medium to log-phase and the localization of Snf7-RFP and Vps13-GFP proteins was observed by confocal microscopy.