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Diverting CERT-mediated ceramide transport to mitochondria triggers Bax-dependent apoptosis.

Journal of cell science (2016-11-27)
Amrita Jain, Oliver Beutel, Katharina Ebell, Sergey Korneev, Joost C M Holthuis
ABSTRACT

A deregulation of ceramide biosynthesis in the endoplasmic reticulum (ER) is frequently linked to induction of mitochondrial apoptosis. Although in vitro studies suggest that ceramides might initiate cell death by acting directly on mitochondria, their actual contribution to the apoptotic response in living cells is unclear. Here, we have analyzed the consequences of targeting the biosynthetic flow of ceramides to mitochondria using a ceramide transfer protein (encoded by COL4A3BP) equipped with an OMM anchor, mitoCERT. Cells expressing mitoCERT import ceramides into mitochondria and undergo Bax-dependent apoptosis. Apoptosis induction by mitoCERT was abolished through (i) removal of its ceramide transfer domain, (ii) disruption of its interaction with VAMP-associated proteins (VAPs) in the ER, (iii) addition of antagonistic CERT inhibitor HPA12, (iv) blocking de novo ceramide synthesis and (v) targeting of a bacterial ceramidase to mitochondria. Our data provide the first demonstration that translocation of ER ceramides to mitochondria specifically commits cells to death and establish mitoCERT as a valuable new tool to unravel the molecular principles underlying ceramide-mediated apoptosis.

MATERIALS
Product Number
Brand
Product Description

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Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
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Anti-Mitochondria Antibody, surface of intact mitochondria, clone 113-1, clone 113-1, Chemicon®, from mouse