Intracellular trafficking of ceramide by ceramide transfer protein

Abstract

The transport and sorting of lipids are fundamental to membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. Ceramide transfer protein (CERT) mediates the ER-to-Golgi trafficking of ceramide. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.

Fig. 1

Biosynthetic pathway of sphingolipids in mammalian cells.

Fig. 2

Redistribution of C₅-DMB ceramide in intact CHO cells. Cells were labeled with 1 μM C₅-DMB ceramide at 4 °C for 30 min, washed and chased at 33 °C for 15 min. For energy inhibition, cells were pretreated with energy inhibitors (50 mM deoxy-d-glucose and 5 mM NaN₃) at 33 °C for 15min, chilled, labeled, and chased in the presence of the energy inhibitors.

Fig. 3

Reconstitution of ER-to-Golgi trafficking of ceramide in perforated cells. (A) The procedure for the reconstitution of ceramide trafficking in semi-intact CHO cells is represented schematically. (B) Reconstitution of ceramide transport from the ER to the Golgi site for SM synthesis in the semi-intact cell system. Perforated wild-type (WT) and LY-A cells pulsed with [³H]sphingosine were chased at 37 °C for 30 min in the transport reaction mixture with the indicated combination of perforated cells (40 μg protein) and cytosolic fraction (100 μg protein). For the cytosol-minus experiments (−), no cytosolic fraction was added to the reaction mixture. The data are expressed as a percentage of the mean value of the wild-type control, where wild-type perforated cells (40 μg protein) were chased in the transport reaction mixture, containing wild-type cytosol (100 μg protein).

Fig. 4

(A) Domains and motifs of CERT. SR, serine-repeat. (B) Regulation of CERT activity. The hyperphosphorylation of SR motif by PKD and CKIγ2 inactivates CERT, and the dephosphorylation by PP2Cɛ activates CERT.

Fig. 5

Crystal structure of the START domain of CERT. (A) The apo-form of the CERT START domain in a ribbon representation. α-Helices (α1-α4), β-strands (β1-β9), and Ω loops (Ω1 and Ω2) are numbered from the N to C terminus. (B) The CERT START domain in a complex with C₁₆-ceramide. The ceramide molecule is represented as space-filling spheres in which yellow, blue, and red spheres represent C, N, and O atoms, respectively.

Fig. 6

A model of CERT-mediated trafficking of ceramide from the ER to the trans Golgi region. SMS, SM synthase; GCS, GlcCer synthase. Inset, efficient trafficking of ceramide through short-distance shuttling by CERT or through the ‘neck-swinging’ movement of the START domain might occur at the sites of contact between the ER and trans Golgi cisternae.

Fig. 7

Structure of a CERT inhibitor.