Identification of TRAMs as sphingolipid-binding proteins using a photoactivatable and clickable short-chain ceramide analog

Abstract

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-C 7 -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-C 7 -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-C 7 -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-C 7 -Cer as a valuable tool for future study of ceramide–protein interactions.

Topological regulation of a transmembrane protein by luminal-to-cytosolic retrotranslocation of glycosylated sequence

“…We detected TM4SF20(A) in untreated cells, and both TM4SF20(A) and TM4SF20(B) in cells treated with C 6 -ceramide ( Figure 1B , upper panel, lanes 3 and 6), a short-chain ceramide analog that is converted to naturally existing long-chain ceramide in cells. 22 , 23 TM4SF20(B) was glycosylated, as treatment with endoglycosidase H (endo H) or peptide: N-glycosidase F (PNGase F) reduced the apparent molecular weight of the protein ( Figure 1B , upper panel lanes 7 and 8). In contrast, TM4SF20(A) was not glycosylated ( Figure 1B , upper panel lanes 3–8).…”

Bifunctional Lipid-Derived Affinity-Based Probes (AfBPs) for Analysis of Lipid–Protein Interactome

“…Over 180 putative sphingolipid-binding proteins were identified by using chemoproteomic profiling; among them seven were further validated. Moreover, Holtuis et al generated ceramide analogues (pacCer and pacGlcCer) with functionalization at the N-linked acyl chain and identified a voltage-dependent anion channel (VDAC2) to be involved in ceramide-mediated apoptosis (Figure E). , Recently, the Ye group reported pac-C7-Cer which contains diazirine and alkyne groups at the sphingosine backbone and found that its interacting protein, translocating chain-associated membrane protein 2 (TRAM2), played an important role in ceramide-induced regulated alternative translocation …”

“…38,39 Recently, the Ye group reported pac-C7-Cer which contains diazirine and alkyne groups at the sphingosine backbone and found that its interacting protein, translocating chain-associated membrane protein 2 (TRAM2), played an important role in ceramide-induced regulated alternative translocation. 40…”

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

“…A few described lipid probes specific for sphingolipids have been published [ 25 , 26 ]. However, no fluorescent ceramide biosensor based on isolated ceramide-binding domains have been described so far, although a range of full-length ceramide-binding proteins have been characterized [ 27 , 28 , 29 , 30 ]. Nevertheless, the localization of these proteins, which are all multi-domain proteins, may be governed by cooperative binding events, including ceramide, but also involving other factors or domains.…”