Dataset Information

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

ABSTRACT: De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.

SUBMITTER: Prashek J

PROVIDER: S-EPMC5572904 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

Prashek Jennifer J Bouyain Samuel S Fu Mingui M Li Yong Y Berkes Dusan D Yao Xiaolan X

The Journal of biological chemistry 20170626 34

<i>De novo</i> synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich ...[more]

PMID: 28652409

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Project description:Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein's involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed 'hydrocarbon chain-first' mechanism for C1P uptake, in contrast to the 'lipid polar headgroup-first' uptake used by most lipid-transfer proteins.

Project description:Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

Dataset Information

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

Publications

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

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