Erythroid Differentiation Dependent Interaction of VPS13A with XK at the Plasma Membrane of K562 Cells

Abstract

Mutations of the bridge-like lipid transport protein VPS13A and the lipid scramblase XK result in Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS), respectively, two similar conditions involving neurodegeneration and deformed erythrocytes (acanthocytes). VPS13A binds XK, suggesting a model in which VPS13A forms a lipid transport bridge between the endoplasmic reticulum (ER) and the plasma membrane (PM), where XK resides. However, studies of VPS13A in HeLa and COS7 cells showed that this protein localizes primarily at contacts of the ER with mitochondria. Overexpression of XK in these cells redistributed VPS13A to the biosynthetic XK pool in the ER but not to PM-localized XK. Colocalization of VPS13A with XK at the PM was only observed if overexpressed XK harbored mutations that disengaged its VPS13A-binding site from an intramolecular interaction. As the acanthocytosis phenotype of ChAc and MLS suggests a role of the two proteins in cells of the erythroid lineage, we explored their localization in K562 cells, which differentiate into erythroblasts upon hemin addition. When tagged VPS13A was overexpressed in hemin-treated K562 cells, robust formation of ER-PM contacts positive for VPS13A was observed and their formation was abolished in XK KO cells. ER-PM contacts positive for VPS13A were seldom observed in undifferentiated K562 cells, despite the presence of XK in these cells at concentrations similar to those observed after differentiation. These findings reveal that the interaction of VPS13A with XK at ER-PM contacts requires a permissive state which depends upon cell type and/or functional state of the cell.

Keywords: contact; endoplasmic reticulum; lipid transfer protein; membrane; plasma membrane.

Figure 1.

Model of VPS13A interaction with XK at the PM as proposed in Guillen-Samander et al. (2022). VPS13A binds VAP in the ER via an FFAT motif located in its N-terminal region, whereas its C-terminal PH domain binds the second cytosolic loop of XK. When explored in fibroblastic cells by exogenous expression of VPS13A and XK, the interaction of the two proteins at ER–PM contacts occurs only when an intramolecular interaction between loop 2 and loop 3 of XK (magnified in inset) is disrupted by point mutations (modified from Guillen-Samander et al., 2022).

Figure 2.

Overexpressed VPS13A is recruited to ER–PM contacts of K562 cells following their differentiation to the erythroid lineage by hemin. (A, B) Equatorial views of live K562 cells expressing VPS13A^Halo and untreated (A) or treated (B) with 30 μM hemin for 2 days and imaged after a brief incubation with TMRE, a live mitochondrial marker. Panel (C) shows the basal plane of the cell shown in the left panels revealing “en face” views of large ER–PM contacts. (D) Quantification of the fraction of PM profiles with VPS13A enrichment (D) (n = 31 untreated cells, 52 hemin-treated cells). (E) Equatorial (left) and basal (right) views of hemin-treated K562 cells co-expressing VPS13A^Halo and GFP-Sec61β. White rectangles outline areas shown at high magnification at the right of the main fields. **p < .01. Scale bars indicate 5 μm.

Figure 3.

Expression of exogenous XK^{KKR > AAA}, but not of exogenous XK^WT, recruits VPS13A to ER–PM contacts also in undifferentiated K562 cells. Cortical regions of untreated or hemin-treated cells co-expressing GFP-XK^WT (A) or GFP-XK^{KKR > AAA} (B) with VPS13A^Halo. PM-associated clusters of VPS13A^Halo and GFP-XK fluorescence reflects ER-PM contacts. (C) Quantification of the fraction of PM with VPS13A (n = 26 untreated XK^WT cells, 46 hemin-treated XK^WT cells, 41 untreated XK^{KKR > AAA} cells, 36 hemin-treated XK^{KKR > AAA} cells). **p < .01, ^n.s.p > 0.05. Scale bars indicate 1 μm.

Figure 4.

Recruitment of overexpressed VPS13A to the PM of erythroid cells depends on XK. Western blot of lysates from COS-7 cells and WT or XK KO K562 cells untreated or treated with hemin for 2 days (A), with quantification of XK intensities normalized to GAPDH input from western blots in (B) (n = 4 untreated, 4 hemin-treated lysates from WT K562 cells). An arrowhead indicates a non-specific band. Western blot of lysates from K562 cells before and after hemin-induced differentiation showing similar levels of VPS13A in the two conditions (C) with quantification of VPS13A intensities normalized to GAPDH (D) (n = 4 untreated, 4 hemin-treated lysates from WT K562 cells). (E–G) Wild-type (E) and XK KO (F) cells untreated or treated with hemin were transfected with VPS13A^Halo. Stippled lines indicate the cell profile in images where such profile is not visible. The fraction of the PM profiles occupied by VPS13A is shown in (G) (n = 23 WT untreated cells, 35 WT hemin-treated cells, 24 XK KO untreated cells, 36 XK KO hemin-treated cells). *p < 0.05, ^n.s.p > 0.05 by a unpaired Student's t-test (G). Scale bars indicate 5 μm.