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Ceramide is a lipid molecule that regulates diverse physiological and pathological reactions in part through inverting the topology of certain transmembrane proteins. This topological inversion is achieved through regulated alternative translocation (RAT), which reverses the direction by which membrane proteins are translocated across the endoplasmic reticulum during translation. However, owing to technical challenges in studying protein–ceramide interaction, it remains unclear how ceramide levels are sensed in cells to trigger RAT. Here, we report the synthesis of pac-C7-Cer, a photoactivatable and clickable short-chain ceramide analog that can be used as a probe to study protein–ceramide interactions. We demonstrate that translocating chain-associated membrane protein 2 (TRAM2), a protein known to control RAT of transmembrane 4 L6 subfamily member 20, and TRAM1, a homolog of TRAM2, interacted with molecules derived from pac-C7-Cer. This interaction was competed by naturally existing long-chain ceramide molecules. We showed that binding of ceramide and its analogs to TRAM2 correlated with their ability to induce RAT of transmembrane 4 L6 subfamily member 20. In addition to probing ceramide–TRAM interactions, we provide evidence that pac-C7-cer could be used for proteome-wide identification of ceramide-binding proteins. Our study provides mechanistic insights into RAT by identifying TRAMs as potential ceramide-binding proteins and establishes pac-C7-Cer as a valuable tool for future study of ceramide–protein interactions. Ceramide is a lipid molecule that regulates diverse physiological and pathological reactions in part through inverting the topology of certain transmembrane proteins. This topological inversion is achieved through regulated alternative translocation (RAT), which reverses the direction by which membrane proteins are translocated across the endoplasmic reticulum during translation. However, owing to technical challenges in studying protein–ceramide interaction, it remains unclear how ceramide levels are sensed in cells to trigger RAT. Here, we report the synthesis of pac-C7-Cer, a photoactivatable and clickable short-chain ceramide analog that can be used as a probe to study protein–ceramide interactions. We demonstrate that translocating chain-associated membrane protein 2 (TRAM2), a protein known to control RAT of transmembrane 4 L6 subfamily member 20, and TRAM1, a homolog of TRAM2, interacted with molecules derived from pac-C7-Cer. This interaction was competed by naturally existing long-chain ceramide molecules. We showed that binding of ceramide and its analogs to TRAM2 correlated with their ability to induce RAT of transmembrane 4 L6 subfamily member 20. In addition to probing ceramide–TRAM interactions, we provide evidence that pac-C7-cer could be used for proteome-wide identification of ceramide-binding proteins. Our study provides mechanistic insights into RAT by identifying TRAMs as potential ceramide-binding proteins and establishes pac-C7-Cer as a valuable tool for future study of ceramide–protein interactions. In addition to being the structural components of membranes, lipids have been increasingly recognized as second messengers for various signal transduction reactions by interacting with their protein sensors. Ceramide is one such lipid that mediates various reactions (1Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. Kelly S. Allegood J.C. Liu Y. Peng Q. Ramaraju H. Sullards M.C. Cabot M. Merrill A.H. Ceramides and other bioactive sphingolipid backbones in health and disease: Lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.Biochim. Biophys. Acta. 2006; 1758: 1864-1884Crossref PubMed Scopus (436) Google Scholar) including cytotoxic and cytostatic effects of chemotherapeutic reagents (2Ogretmen B. Sphingolipid metabolism in cancer signalling and therapy.Nat. Rev. Cancer. 2018; 18: 33-50Crossref PubMed Scopus (459) Google Scholar). Among these reagents, doxorubicin blocks proliferation of cancer cells by stimulating proteolytic activation of cAMP response element–binding protein 3-like 1 (CREB3L1), a membrane-bound transcription factor (3Ye J. Transcription factors activated through RIP (regulated intramembrane proteolysis) and RAT (regulated alternative translocation).J. Biol. Chem. 2020; 295: 10271-10280Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 4Denard B. Lee C. Ye J. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1.ELife. 2012; 18e00090Google Scholar, 5Denard B. Pavia-Jimenez A. Chen W. Williams N.S. Naina H. Collins R. Brugarolas J. Ye J. Identification of CREB3L1 as a biomarker predicting doxorubicin treatment outcome.PLoS One. 2015; 10e0129233Crossref PubMed Scopus (15) Google Scholar). In the absence of doxorubicin, CREB3L1 exists as an inactive transmembrane precursor, as cleavage of the protein is inhibited by transmembrane 4 L6 subfamily member 20 (TM4SF20), a polytopic transmembrane protein (6Chen Q. Lee C.-E. Denard B. Ye J. Sustained induction of collagen synthesis by TGF-β requires regulated intramembrane proteolysis of CREB3L1.PLoS One. 2014; 9e108528Crossref PubMed Scopus (36) Google Scholar). Doxorubicin induces production of ceramide (4Denard B. Lee C. Ye J. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1.ELife. 2012; 18e00090Google Scholar), which in turn reverses the direction through which the first transmembrane helix of TM4SF20 is translocated across the Sec61 translocon in the endoplasmic reticulum (ER) through a process designated as regulated alternative translocation (RAT) (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). As a result, the topology of newly synthesized TM4SF20 produced under this circumstance (referred as TM4SF20(B)) is completely opposite to that generated in the absence of ceramide (referred as TM4SF20(A)) (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). This topological inversion turns TM4SF20 from an inhibitor to an activator for proteolysis of CREB3L1 (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), a reaction enabling translocation of the N-terminal domain of CREB3L1 from membranes to the nucleus, where it activates transcription of genes that inhibit cell proliferation (4Denard B. Lee C. Ye J. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1.ELife. 2012; 18e00090Google Scholar, 8Denard B. Seemann J. Chen Q. Gay A. Huang H. Chen Y. Ye J. The membrane-bound transcription factor CREB3L1 is activated in response to virus infection to inhibit proliferation of virus-infected cells.Cell Host Microbe. 2011; 10: 65-74Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Sphingolipids are also involved in inhibition of macrophage chemotaxis mediated by chemokine receptors including C–C chemokine receptor 5 (CCR5), a G protein–coupled receptor (9Eberle M. Ebel P. Mayer C.A. Barthelmes J. Tafferner N. Ferreiros N. Ulshöfer T. Henke M. Foerch C. de Bazo A.M. Grösch S. Geisslinger G. Willecke K. Schiffmann S. Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils.Immunol. Cell Biol. 2015; PubMed Scopus Google Scholar, B. Han S. J. Ye J. G receptors by topological PubMed Scopus Google Scholar). production of in which in turn the topology of newly synthesized through RAT. with the topology as a chemokine topological inversion of in part are to chemotaxis B. Han S. J. Ye J. G receptors by topological PubMed Scopus Google Scholar, E. molecules and Full Text Full Text PDF PubMed Scopus Google Scholar). that topological inversion of certain transmembrane proteins through RAT is for of the signal However, it remains unclear how the are sensed in cells in to trigger RAT. We that translocation chain-associated membrane protein 2 (TRAM2), a homolog of that is known to be a Sec61 protein E. S. protein of the endoplasmic reticulum involved in PubMed Scopus Google Scholar, H. J. The of membrane proteins into the is a Full Text Full Text PDF PubMed Scopus Google Scholar), is involved in RAT of TM4SF20 (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). proteins a domain that is also in ceramide which is to E. and of a of Full Text Full Text PDF PubMed Scopus Google Scholar). However, a interaction proteins and ceramide been In the we the interaction through a photoactivatable and clickable analog of short-chain ceramide we Our study that TRAMs be the sphingolipid that regulates translocation of certain transmembrane proteins. to study reactions is that ceramide is in it to cells with the In a ceramide-binding proteins by cells with a photoactivatable and clickable analog of which is to ceramide in cells by a reaction by ceramide P. B. J. C. for of Chem. Biol. 2016; PubMed Scopus Google Scholar, P. H. A. M. T. M. B. J.C. with and in Full Text Full Text PDF PubMed Scopus Google Scholar). However, was to the ceramide that as cells with to induce RAT of TM4SF20 to the topology of which is opposite to that of the protein produced in the absence of ceramide 4 and In to cells with a ceramide analog a RAT of TM4SF20 to as (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The of is that of the inversion potential from to the where can be (7Chen Q. Denard B. Lee C.-E. Han S. Ye James S. Ye J. Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix.Mol. Cell. 2016; 63: 567-578Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). is to naturally existing long-chain ceramide through reactions by and ceramide in cells K. J. The sphingolipid in ceramide metabolism and PubMed Scopus Google Scholar). this is to RAT of the of the in the ceramide analog be to induce this as it be and by naturally existing long-chain was as as in stimulating RAT of TM4SF20 short-chain trigger signaling reactions that be by we to a photoactivatable and clickable analog of to ceramide-binding proteins. analysis, we that pac-C7-Cer was to long-chain and its in and this was inhibited by treatment with a ceramide synthase inhibitor that blocks of short-chain to long-chain B. R. J. A. of the of ceramide in response to ceramide in the cell for ceramide in the of Biol. Chem. 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Lee C.-E. Denard B. Ye J. Sustained induction of collagen synthesis by TGF-β requires regulated intramembrane proteolysis of CREB3L1.PLoS One. 2014; 9e108528Crossref PubMed Scopus (36) Google Scholar), in of and and in in a proteins to membranes and with the protein as J. A. and by in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google produced from from the a generated in this study by mice with to and of and TRAM2 a generated in this study by with to of with a the of by was as C. C. K. A. M. K. J. A. A. metabolism by Chem. Biol. 2012; PubMed Scopus Google Scholar) that the was 6 and under an with a and from as for the for under in a with and in in with to protein was to reactions to cell was with in of 1 in 1 in and in The was for 1 The reaction in protein was by of the reaction with of for the with in in and with with and for The with in and with in The proteins from the by in for 20 Sphingolipids by in as (4Denard B. Lee C. Ye J. 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Deng et al. (Mon,) studied this question.
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