Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides

Yong-Guang Gao,Xiuhong Zhai,Ivan A Boldyrev,Julian G Molotkovsky,Dinshaw J Patel,Lucy Malinina,Rhoderick E Brown

doi:10.1016/j.jbc.2021.100600

Abstract

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.

Highlights

Lipid intracellular transport by vesicular and nonvesicular mechanisms helps maintain distinct lipid compositions associated with various cell organelles
To determine whether Ceramide-1-phosphate transfer proteins (CPTPs) and related glycolipid transfer protein (GLTP) homologs contain targeting motifs for specific phosphoglycerides embedded in membranes, we investigated the regulatory effects exerted by various phosphoinositides (PIPs) on SL transfer by CPTP, accelerated cell death-11 protein (ACD11), or GLTP and their membrane partitioning
We focused on PIPs present in the trans-Golgi and plasma membrane (phospatidylinositol-4,5-bisphosphate; PI-(4,5)P2) due to earlier findings of CPTP enrichment at these intracellular sites [22]

Summary

Introduction

Lipid intracellular transport by vesicular and nonvesicular mechanisms helps maintain distinct lipid compositions associated with various cell organelles. LTPs involved in the nonvesicular trafficking of sphingolipids (SLs) between membranes include ceramide transfer protein (CERT) [13, 14], certain SL activator proteins [15,16,17,18], and members of the glycolipid transfer protein (GLTP) superfamily [6,7,8, 19,20,21]. GLTP superfamily members such as CPTP, ACD11, and GLTP lack known lipid-binding domains (LBDs) (e.g., PH, PZ, C1, C2) that target various proteins to select phosphoglycerides in intracellular membranes [37,38,39,40,41,42,43]. The data are consistent with PIP-specific headgroup interaction sites existing on CPTP but not GLTP that serve a dual role of enhancing SL transfer activity while acting as preferred targeting/docking sites in specific membranes in vivo. Mapping of the PIP-selective motifs in C1P-specific GLTP-folds within membrane interaction regions reveals a role for the recently discovered ID-loop (α3-α4 helices connecting loop) [27]

Results

Discussion

Conclusion