Proteome‐wide Identification of Ceramide‐binding Proteins Reveals TRAM Proteins as Sphingolipid Sensors That Regulate Translocation of Transmembrane Proteins

Ceramide are central intermediates of sphingolipid metabolism, mediating cell proliferation, mitochondrial apoptosis, and intracellular trafficking. Ceramide regulates diverse physiological and pathological reactions in part through inverting 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. We previously reported that RAT of transmembrane 4 L6 subfamily member 20 (TM4SF20) induced by ceramide converts the topology of TM4SF20. As a result, TM4SF20 with the different topology triggers proteolytic activation of a transcription factor called cAMP responsive element binding protein 3‐like 1 (CREB3L1), which in turn activates transcription of genes that inhibit cell proliferation. However, it remains unclear how ceramide levels are sensed in cells to trigger the ceramide‐induced signaling reactions including RAT. Here we describe a chemoproteomic strategy that uses photo‐activatable and clickable short chain ceramide (pac‐C7‐Cer) in combination with stable isotope labeling by amino acids in cell culture (SILAC)‐based mass spectrometry to perform a proteome‐wide identification of ceramide‐binding proteins directly in living cells. This approach yields 24 ceramide‐binding proteins repeated in three independent experiments. Among these proteins, we determine that translocating chain associated membrane protein 1 (TRAM1), an ER translocon‐associated proteins, and TRAM2, a protein known to be involved in RAT of TM4SF20, are specific ceramide‐binding proteins. We address that interrupting the binding of TRAM proteins to ceramide derived from pac‐C7‐Cer by inhibiting the conversion of short chain ceramide to long chain ceramide will also inactivate RAT of TM4SF20 induced by C6‐ceramide. Furthermore, we demonstrate that B13, by serving as a competitive inhibitor that blocked binding of ceramide to TRAM proteins prevented ceramide‐induced RAT of TM4SF20. It suggests that TRAM proteins might be the ceramide sensor involved in RAT regulation. Above all, this study supports a role of TRAM proteins in RAT regulation induced by ceramide and provides a molecular framework for understanding of global cellular function exerted by sphingolipids interacting proteins.