A Role of Phosphatidylserine in the Function of Recycling Endosomes

Abstract

Cells internalize proteins and lipids in the plasma membrane (PM) and solutes in the extracellular space by endocytosis. The removal of PM by endocytosis is constantly balanced by the replenishment of proteins and lipids to PM through recycling pathway. Recycling endosomes (REs) are specific subsets of endosomes. Besides the established role of REs in recycling pathway, recent studies have revealed unanticipated roles of REs in membrane traffic and cell signalling. In this review, we highlight these emerging issues, with a particular focus on phosphatidylserine (PS), a phospholipid that is highly enriched in the cytosolic leaflet of RE membranes. We also discuss the pathogenesis of Hermansky Pudlak syndrome type 2 (HPS2) that arises from mutations in the AP3B1 gene, from the point of view of dysregulated RE functions.

Keywords: bioID proximity labeling; endosomes; flippase; membrane traffic; phosphatidylserine; pleckstrin-homology domain.

FIGURE 1

PS is enriched in REs, an organelle that serves as a hub for a variety of membrane traffic. slow recycling: PM→ EEs→ REs→ PM; retrograde transport: PM→ EEs→ REs→ the Golgi; exocytic transport: ER→ the Golgi→ REs→ PM. PS is concentrated in the cytosolic leaflet of RE membranes. Chemical structure of PS is shown in the right. The headgroup of PS (phosphoserine, shown in red) has one net negative charge.

FIGURE 2

evectin-2 PH specifically binds to PS (A) In vitro lipid-binding assays. His-tagged evectin-2 PH was mixed with liposomes composed of brain phosphatidylcholine (PC), brain phosphatidylethanolamine (PE), and the indicated negatively charged lipid (20% mol/mol of total lipids). The mixture was then spun at 100,000 g, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE, followed by Coomassie blue staining (B) Overall structure of human evectin-2 PH in complex with phosphoserine, the headgroup of PS (C) Charge distribution surface model of evectin-2 PH in complex with phosphoserine (stick model). The surface is colored according to the electrostatic potential of the residues (blue, positive; red, negative). Hydrophobic residues around the PS-binding pocket, which are expected to be inserted into the membrane, are indicated. Data were reproduced and modified from (Uchida et al., 2011).

FIGURE 3

Two methods to examine intracellular PS distribution with 2xPH (Left) 2xPH tagged with a fluorescent protein, such as EGFP, is expressed in the cytosol by plasmid transfection. In this case, 2xPH detects PS in the cytosolic leaflet of the PM and organelle membranes (Right) Recombinant 2xPH detects PS in both leaflets when applied to fixed and permeabilized cells. 2xPH can be detected by immunocytochemistry using antibodies against the tag attached to 2xPH.

FIGURE 4

ATP8A1 regulates membrane trafficking and signalling at REs. ATP8A1 flips PS to the cytosolic leaflet of RE membranes. The PS then recruits a membrane fission protein EHD1 from the cytosol to REs. The EHD1-mediated fission of RE membranes generates membrane transport carriers. PS in REs also facilitates the nuclear translocation of YAP, thereby promoting the transcription of YAP-target proliferative genes, such as CTGF (connective tissue growth factor).

FIGURE 5

Deficiency of AP-3 results in YAP activation. AP-3 mediates ATP8A1 transport from endosomes to lamella bodies in alveolar type 2 cells. In AP-3-knockout cells, because the trafficking of ATP8A1 from endosomes is impaired, ATP8A1 is forced to re-localize to REs, thereby increasing the levels of cytosolic PS in RE membranes. The PS enrichment in RE membranes promotes aberrant activation of YAP, which augments cell proliferation and migration.