Structural Basis for the Regulation of the Ceramide Transfer Protein
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Ceramide, a central intermediate in sphingolipid metabolism, is synthesized at the endoplasmic reticulum (ER) and transferred to the Golgi by the ceramide transfer (CERT) protein for conversion to sphingomyelin (SM). CERT is a multi-domain protein that contains an amino terminal pleckstrin homology (PH) domain and a carboxy terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. PH targets CERT to the Golgi through interactions with phosphatidylinositol-4-monophosphate (PtdIns(4)P) and START carries out the transfer of ceramide. Following PH is a short serine rich (SR) motif. Phosphorylation of SR inhibits both PH and START function, but only when both are present on the same molecule, suggesting an auto-inhibitory interaction. In this study, we used biochemical and structural techniques to investigate the role of the PH and START domains and their interactions in the function and regulation of CERT. Our structural studies showed that CERT PH binds to PtdIns(4)P via the canonical ligand binding pocket of PH domains. With a better understanding of PH, we turned our focus to the PH/START interaction. Employing a combination of structural techniques, we determined that PH binds START at the same binding interface used to bind PtdIns(4)P. Consistent with the structural findings, START was shown to inhibit PH/PtdIns(4)P binding in vitro. To determine how this interaction regulates full-length CERT, we characterized the function of CERT mutants with disrupted PH/START interactions. Our results revealed that the PH/START auto-inhibitory interaction reduces the PtdIns(4)P binding, ceramide transfer activity, and Golgi localization of CERT. The results also indicated the PH/START interaction occurs in both SR phosphorylated and dephosphorylated CERT. We propose the following model for CERT regulation. The PH and START domains interact and inhibit dephosphorylated CERT until PH binding to the PtdIns(4)P-enriched Golgi disrupts the PH/START interaction. However, following SR phosphorylation, the PH/START interaction can no longer be disrupted by PtdIns(4)P binding and CERT remains inactive. Studies in this dissertation provide important insights into CERT function and regulation.
Table of Contents
Introduction -- Materials and methods -- Structural basis for the binding of PtdIns(4)P by the PH domain of CERT -- Interaction between the PH and start domains of CERT competes with PtdIns(4)P binding by the PH domain -- PtdIns(4)P binding releases the inhibition of CERT -- Final discussion
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Ph.D.