© 2015 Nature America, Inc. All rights reserved. NATURE CHEMICAL BIOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology 1 ARTICLE PUBLISHED ONLINE: 17 AUGUST 2015 | DOI: 10.1038/NCHEMBIO.1889 I n mammalian cells, sphingomyelin (SM) is the most abundant sphingolipid and a reservoir for generating the second messenger ceramide. The LEs are a major site for sphingolipid catabolism. In LEs, ceramide can be generated by acid sphingomyelinase (SMPD1) hydrolyzing SM on the intraluminal vesicles (ILVs) of multivesic- ular bodies (MVBs) 1,2 . Ceramide can be further degraded by acid ceramidase (ASAH1), releasing a fatty acid and sphingosine, the final breakdown products in LE sphingolipid catabolism. The mech- anism by which sphingosine exits LEs is not fully understood 3–5 . However, sphingosine can enter the salvage pathway to be used as a building block for the regeneration of SM. In this pathway, sphingosine is initially converted to ceramide in the endoplasmic reticulum (ER). The formed ceramide can then be transferred by ceramide transfer protein (CERT) to the trans-Golgi, where it is used as a substrate for SM synthesis 6 . The sphingolipid metabolic pathways are attracting increasing attention as targets for anticancer strategies 7–9 . Ceramide is a strong tumor suppressor with proapoptotic properties, and several com- monly used chemotherapeutics induce cell death in a ceramide- dependent fashion 10,11 . Cancer cells often downregulate SMPD1 to reduce their ceramide content and increase chemotherapy resis- tance 12 . Consequently, SM may accumulate in LEs, leading to lyso- somal destabilization 13,14 . Because of this, SMPD1 manipulation and lysosomal membrane permeabilization (LMP) are being explored as means to kill chemotherapy-resistant cancer cells 15,16 . Importantly, both the subcellular localization and the abundance of ceramide are relevant. In particular, ceramide and its metabolites at ER– mitochondrial membrane contact sites promote mitochondrial outer membrane permeabilization, cytochrome C release and caspase- dependent apoptosis 17–19 . Accordingly, ceramide retention in the ER by inhibition of CERT can sensitize cells to chemotherapeutics 20 . LAPTM4B belongs to the membrane-spanning lysosomal LAPTM family of proteins and is aberrantly expressed in several common cancers 21–25 . LAPTM4A, the first member of the LAPTM protein family to be described, was characterized as a nucleoside transporter 26 and later found to confer multidrug resistance by modulating drug compartmentalization 27,28 . Similarly, LAPTM4B has been reported to affect the subcellular distribution of cyto- toxic drugs 23,29 , but endogenous substrates for LAPTM4B-mediated transport have not been identified. High LAPTM4B expression is associated with resistance to anthracyclines, possibly promoting cytosolic retention of the drugs and thereby reducing DNA dam- age 23 . LAPTM4B has also been reported to stimulate the efflux of chemotherapeutic compounds from cells through the P-glycoprotein efflux pump 29 and to promote lysosomal membrane stability 21 . However, in a large survey of four patient data sets, low expression of LAPTM4B was strongly associated with chemotherapy resistance 25 . Very recently, LAPTM4B was found to regulate epidermal growth factor receptor signaling by acting on its lysosomal sorting and degradation 30 and autophagy initiation 31 . Thus, LAPTM4B appears to be relevant for cancer progression by several mechanisms, and high or low LAPTM4B expression may confer a poor anticancer drug response. We recently developed a strategy to follow LE SM catabolism and recycling by tracking the metabolism of 3 H-labeled SM tar- geted to the LE in low-density lipoprotein (LDL) particles 5 . Using this assay, we conducted a small interfering RNA (siRNA) screen to find proteins that facilitate the removal of SM degradation products from the LE. LAPTM4B was identified as the best hit in this screen. Here we provide evidence that LAPTM4B binds ceramide and that the amount of LAPTM4B in the LE membrane is a major determi- nant of intra-endosomal sphingolipid content and thereby affects LE membrane stability. Moreover, LAPTM4B acts as a gatekeeper between intra- and extra-endosomal ceramide pools, modulating apoptosis sensitivity. Thus, by regulating the subcellular compart- mentalization of ceramide, LAPTM4B controls key sphingolipid- mediated cell death mechanisms that are highly relevant in cancer. RESULTS LAPTM4B deficiency causes cellular ceramide accumulation To identify regulators of LE sphingolipid export, we screened 19 candidate membrane-spanning LE proteins (annotated by 1 Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 2 Minerva Foundation Institute for Medical Research, Helsinki, Finland. 3 Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York, USA. 4 Department of Biochemistry, University of Geneva, CH-1211 Geneva 4, Switzerland. *e-mail: tomas.blom@helsinki.fi or elina.ikonen@helsinki.fi LAPTM4B facilitates late endosomal ceramide export to control cell death pathways Tomas Blom 1,2 *, Shiqian Li 1,2 , Andrea Dichlberger 1,2 , Nils Bäck 1 , Young Ah Kim 3 , Ursula Loizides-Mangold 4 , Howard Riezman 4 , Robert Bittman 3 & Elina Ikonen 1,2 * Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physio- logical function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and indepen- dent of acid ceramidase–dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid- mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.