César Botella
Function of a P4-type ATPase in the homeostasis of membrane glycolipids in Arabidopsis thaliana
Published on 7 December 2016
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Thesis presented December 07, 2016
Abstract:
In a eukaryotic cell, each membrane compartment has a specific lipid composition, regulated according to physiological and environmental conditions. This lipid homeostasis results from coordination of lipid synthesis, conversion, degradation and trafficking. Whereas most enzymes involved in lipid metabolism are now identified, most steps of lipid transport remain to be characterized. We focus on the chloroplast lipid homeostasis, particularly on galactolipid homeostasis, essential lipids of photosynthetic membranes. This lipids are synthetized within the chloroplast's envelope. However, a majority of galactolipids derived from phosphatidylcholine which is synthetized in the endoplasmic reticulum. The delocalization of this synthesis pathway underline the importance of the associated lipid trafficking. Transcriptomic studies have highlighted that ALA10 is overexpressed in condition modifying the galactolipids synthesis such as the chemical inhibition of MGDG synthesis by the galvestine-1, or during phosphate starvation.
The aim of this thesis is to characterize ALA10, analyzing its role concerning this lipid trafficking and the chloroplastic galactolipid homeostasis.
To understand ALA10's role, we firstly have used GFP fusion to determine its subcellular localization and analyzed lipid composition of different plant lines expressing ALA10 at different levels. Lipid analysis show that ALA10 boosts galactolipid synthesis and limits endoplasmic reticulum located phosphatidylcholine desaturation. We searched ALA10's potential partners in order to explain this effects and studied their interaction using a bimolecular fluorescence complementation approach. We determined that ALA10 interacts with ALIS1 and ALIS5, two beta subunit potentially necessary for ALA10's localization and function, and confirmed the colocalization of these proteins with ALA10 using GFP/CFP fusions. ALA10 with ALIS5 can localize within the endoplasmic reticulum in close proximity to chloroplast, or near the plasma membrane with ALIS1. We have also determined that ALA10 interacts with a fatty acid desaturase, FAD2 and with an E3-ubiquitine ligase PUB11. FAD2 interaction confirms the link between ALA10 and phosphatidylcholine desaturation. Then we have studied the ALIS1 and ALIS5 effect on ALA10 function using KO lines for these proteins or overexpressor lines in conjunction with ALA10 overexpression. Electron microscopy observation revealed that the chloroplast morphology and their relations with endomembrane system are modified depending of the ALIS expressed with ALA10. Lipid analysis on KO lines confirms an impact of ALA10 on galactolipids homeostasis as well as in phosphatidylcholine desaturation. This effect appears to be variable depending of the photoperiod or the circadian rhythm indicating a post traductional regulation of ALA10. The role of PUB11 in this regulation have been studied.
Finally this study reveal that, in chlorophyll cells, the endoplasmic reticulum phospholipid flippase ALA10 is involved in the desaturation process of phosphatidylcholine. Its activity stimulates galactolipid synthesis and activates biogenesis of photosynthetic membranes, probably by promoting lipids exchange between chloroplasts and endoplasmic reticulum.
Keywords:
flippase, P4 ATPase, lipid trafficking, plant physiology
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