Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments.

Yoko Ito,Yvon Jaillais,Louise Fougère,Fabrice Cordelières,Nicolas Esnay,Yohann Boutté,Matthieu Pierre Platre

doi:10.3390/ijms22168450

Abstract

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.

Highlights

In eukaryotic cells, cellular functions are extensively supported by membrane trafficking which sorts and targets proteins and lipids into distinct destinations
It has recently been observed that the adaptor protein 4 (AP4) labels a sub-domain of the trans-Golgi network (TGN) that co-localizes with the SNARE protein VAMP727 that is involved in vacuolar trafficking via multivesicular bodies (MVBs) while AP1 labels a sub-domain of the TGN that co-localizes with the SNARE protein VAMP721 that functions in secretory trafficking to the plasma membrane [17,18,19,20]
Our results show that very long chain fatty acid (VLCFA), which are predominant in the pool of sphingolipids, are involved in the homeostasis of PI3P at endosomal compartments

Summary

Introduction

Cellular functions are extensively supported by membrane trafficking which sorts and targets proteins and lipids into distinct destinations. It was suggested that the pre-vacuolar compartment MVBs mature from the TGN; this has been supported by electron microscopy observations of localization of Endosomal Sorting Complex Required for Transport (ESCRT I to III) and pharmacological treatment that inhibits MVB formation [21]. In Arabidopsis, fluorescent biosensors have revealed that PI3P is mainly localized to MVBs and vacuoles with a sparse pool are present at VHA-a1-positive sub-domain of the TGN [25,26]. While the quantification of PI3P levels at the compartments during the association time was technically too challenging to address in this study, we used a pharmacological approach to suggest that the pool of PI3P at the TGN could be transferable to MVBs. Altogether, our results show that VLCFAs, which are predominant in the pool of sphingolipids, are involved in the homeostasis of PI3P at endosomal compartments

Results

Plant Material and Growth Conditions

Inhibitor Treatments

Confocal Microscopy and Image Analyses

Statistical Analyses