RESUMEN
Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.
Asunto(s)
Glucosilceramidas , ATPasas Tipo P , Humanos , Glucosilceramidas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Adenosina Trifosfatasas/metabolismo , Homeostasis , ATPasas Tipo P/metabolismoRESUMEN
Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.
Asunto(s)
Enfermedad de Gaucher , Humanos , Enfermedad de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Fibroblastos/metabolismo , Lisosomas/metabolismo , Membranas Intracelulares/metabolismoRESUMEN
ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.
Asunto(s)
Adenosina Trifosfatasas , Polaridad Celular , Membrana Celular , Citoplasma , Proteínas del Citoesqueleto , FosfoproteínasRESUMEN
ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.