ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells

Abstract

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca2+-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4BE235Q, a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP–Rab5AQ79L). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.

Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors

“…As shown in Figure 1 , intensity of the WB signal of Scr3 in the TCL was slightly stronger in 3T3 cells than in 3T3-L1 cells under both subconfluent ( sub ) and confluent ( con ) conditions despite weaker or similar signal intensities of the two loading controls used ( flotillin-2 and GAPDH ). We previously found that Scr3, either exogenously expressed in human embryonic kidney (HEK)293 cells or endogenously expressed in human bladder carcinoma T-24 cells, was consistently secreted into the culture medium in the form of extracellular microvesicles (exosomes) [ 18 ]. Likewise, secretion of Scr3 as the form of exosomes was also observed in mouse 3T3 and 3T3-L1 cells ( Figure 1 ).…”

“…Decrease in Scr3 in the protein level (to 13.9–22.6%) more than in the mRNA level (to 33.2–54.0%) during adipocyte differentiation is partly explained by enhanced secretion of Scr3 in the form of extracellular microvesicles named exosomes ( Figures 1 – 3 and Supplementary Figure S2). We previously reported that exosomal secretion of exogenously expressed Scr3 was significantly reduced and that subcellular localization was changed from the cytoplasmic puncta to nucleus by inhibition of palmitoylation of Scr3 by 2-bromopalmitate in HEK293 cells [ 18 ]. Since 2-bromopalmitate was toxic to 3T3-L1 cells when the cells were cultured for a long time, we could not investigate the effects of inhibition of palmitoylation of Scr3 on adipogenesis.…”

“…TUB and TULPs bind phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and are liberated from the plasma membrane upon activation of G-protein-coupled receptors [ 15 , 16 ]. It is still not clear whether the topology of the C-terminal α-helical regions of PLSCRs is a transmembrane helix [ 4 , 17 ] or a helix that fills the central hole of the β-barrel [ 13 , 18 ]. However, PLSCRs have been shown to contain functional non-classical nuclear localization signals [ 19 , 20 ], and thus the nuclear translocation of PLSCRs by treatment with a palmitoylation inhibitor (2-bromo-palmitate) or by palmitoylation site mutation favour the latter hypothesis at least in vivo [ 18 , 21 ].…”

Membrane lipid interactions in intestinal ischemia/reperfusion-induced Injury

“…While it is important to point out the different cell types under investigation, neurons in the Rami study and endothelial cells in ours, both suggest an increase in PLSCR1 shortly after reperfusion is established. As for the decrease in PLSCR1 protein during the hypoxic period that we observed, the potential for PLSCR1 to be released from the cell membrane must be considered and may be due to shedding of the protein into the culture media as was recently described for PLSCR3 [146]. …”

Roles and regulation of phospholipid scramblases

“…For example, mutagenesis of the cysteine residues of PLSCR3 critical for its palmitoylation led to its redistribution to the nuclear compartment [85]. Furthermore, secretion of PLSCR3 in exosomes was inhibited by 2-bromopalmitate [86]. NLS signals are also found in PLSCR2, PLSCR3, and PLSCR4.…”