ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells.
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 VPS4B(E235Q), 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-Rab5A(Q79L)). 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.
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Tatsutoshi
T
Inuzuka
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Akira
A
Inokawa
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Cen
C
Chen
†Department of Food and Nutrition, Kyoto Women's University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan.
Kumiko
K
Kizu
†Department of Food and Nutrition, Kyoto Women's University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan.
Hiroshi
H
Narita
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Hideki
H
Shibata
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Masatoshi
M
Maki
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D055354:Exosomes / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation / Q000187:drug effects*; D010168:Palmitates / Q000494:pharmacology; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; C097753:PDCD6 protein, human; C493424:PLSCR3 protein, human; D010168:Palmitates; D049294:Phospholipid Transfer Proteins; D049452:Green Fluorescent Proteins; C022776:2-bromopalmitate