Efficient Trafficking of Ceramide from the Endoplasmic Reticulum to the Golgi Apparatus Requires a VAMP-associated Protein-interacting FFAT Motif of CERT

TLDR

Ceramide is synthesized in the ER and must be transported to the Golgi for conversion to sphingomyelin, a process mediated by CERT, which contains a PH domain for Golgi targeting, a START domain for ceramide transfer, and an FFAT motif that binds ER‑resident VAP proteins. Mutations disrupting CERT’s FFAT motif abolish its interaction with VAP‑A/B and impair ER‑to‑Golgi ceramide transport in intact cells, although they do not affect Golgi targeting or START‑domain transfer activity in vitro, indicating that the PH domain and FFAT motif spatially constrain CERT’s ceramide‑transfer function.

Abstract

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.