A partnership between the lipid scramblase XK and the lipid transfer protein VPS13A at the plasma membrane

Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER–PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.

Chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS) are two similar diseases, characterized by progressive degeneration of the caudate nucleus leading to chorea and other movement defects and by abnormally shaped erythrocytes (acanthocytes). Chorea-acanthocytosis is due to loss of function of VPS13A (1, 2), the founding member of a superfamily of lipid transport proteins, also called chorein motif proteins because of the high conservation of their most N-terminal region (∼120 amino acids [aa]), referred to as the chorein motif. Members of this superfamily, which also comprises the autophagy factor ATG2, are localized at membrane contact sites where they are thought to transfer lipids unidirectionally between adjacent bilayers of different organelles by a bridge-like mechanism (3–5). McLeod syndrome is due instead to loss of function of XK (6), a member of a family of lipid scramblases whose function is to collapse the heterogeneity of the lipid composition of the two leaflets of the plasma membrane (PM) (7). A consequence of this scrambling is the exposure to the cell surface of PtdSer, a phospholipid that is recognized by phagocytic cells and normally concentrated in the cytosolic leaflet of the PM by the action of flippases (8).Consistent with the similarity of the clinical conditions resulting from mutations in VPS13A and in XK, there is evidence suggesting that the two proteins are functional partners (9–11). Thus, it is of great interest that both proteins are implicated in lipid transport. More importantly, as chorein motif proteins do not penetrate lipid bilayers, but are thought to achieve net transfer of lipids between cytosolic leaflets of the donor and receiving membranes, they are expected to require the cooperation of scramblases to allow equilibration of lipid mass between the bilayer leaflets. Accordingly, there is evidence for the partnership of the chorein motif protein ATG2 with a lipid scramblase in the growth of the autophagosome membrane (4, 12, 13). Cooperation between VPS13A and XK, which has recently been confirmed by in vitro studies to have scramblase activity (14), would represent another example of such a partnership, providing clues to mechanisms of disease in chorea-acanthocytosis and McLeod syndrome. Strong support for this possibility came by the demonstration of an interaction between these two proteins: 1) studies of McLeod syndrome erythrocytes revealed that lack of XK results in a loss of VPS13A in their membranes (9), as expected if VPS13 and XK are part of the same complex; 2) their interaction was supported by biochemical experiments (9–11); and 3) the localization of overexpressed VPS13A at contacts between the endoplasmic reticulum (ER) and lipid droplets was shown to be abolished by cooverexpression of XK, resulting in a localization of VPS13A along with XK throughout the ER (10).So far, however, there has been no evidence from imaging studies in mammalian cells that VPS13A colocalizes with XK at the PM, where XK is expected to act. Studies in HeLa cells with tagged-VPS13A—both overexpressed VPS13A (10, 15–17) and VPS13A expressed at endogenous levels (15)—have shown that this protein is localized at contacts between the ER and mitochondria and lipid droplets. These studies have further shown that this localization of VPS13A is mediated by the interaction of its N-terminal region with the MSP domain of the ER protein VAP and of its C-terminal region with a yet to be defined binding site on mitochondria and on lipid droplets (15, 17). An interaction of VPS13A with mitochondria was also supported by the identification of this protein as a hit in a screen for mitochondria neighbors by proximity biotinylation (18, 19). Based on these findings, it has been proposed that VPS13A, like its paralog VPS13D, mediates transport of lipids to mitochondria, which are organelles not connected by vesicular transport to the ER, where most membrane lipids are synthesized (15, 20).The goal of this study was to determine how VPS13A interacts with XK and to determine whether such interaction can occur at the PM.