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Subcellular localization of sphingomyelin revealed by two toxin‐based probes in mammalian cells
Author(s) -
Yachi Rieko,
Uchida Yasunori,
Balakrishna Bhat Hema,
Anderluh Gregor,
Kobayashi Toshihide,
Taguchi Tomohiko,
Arai Hiroyuki
Publication year - 2012
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2012.01621.x
Subject(s) - sphingomyelin , endosome , biology , intracellular , microbiology and biotechnology , gene knockdown , organelle , biochemistry , phospholipid , membrane , gene
Sphingomyelin ( SM ) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS ‐1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin‐ II ( E qt II ). Both toxins stained SM in the plasma membrane ( PM ), and the stains were abolished by sphingomyelin synthase 2 ( SMS 2) knockdown or sphingomyelinase ( SM ase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by E qt II , and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes ( LE s) among intracellular organelles, whereas E qt II stained recycling endosomes ( RE s) in addition to LE s. The intracellular SM stains by E qt II were abolished by sphingomyelin synthase 1 ( SMS 1) knockdown, but not by SMS 2 knockdown. These results indicate that lysenin and E qt II label different SM pools and that SMS 2 and SMS 1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS ‐1 cells. The use of the two SM ‐binding probes may provide more insights into various sphingomyelin‐mediated processes in different topological domains.