PUB11-Dependent Ubiquitination of the Phospholipid Flippase ALA10 Modifies ALA10 Localization and Affects the Pool of Linolenic Phosphatidylcholine

Abstract

Biogenesis of photosynthetic membranes depends on galactolipid synthesis, which relies on several cell compartments, notably the endoplasmic reticulum (ER) and the chloroplast envelope. Galactolipid synthesis involves lipid trafficking between both membrane compartments. In Arabidopsis, ALA10, a phospholipid flippase of the P 4 type-ATPase family, counteracts the limitation of monogalactosyldiacylglycerol (MGDG) production and has a positive effect on leaf development. ALA10 locates in distinct domains of the ER depending on the ALIS (ALA interacting subunit) subunit it interacts with: close to the plasma membrane with ALIS1, or next to chloroplasts with ALIS5. It interacts with FAD2 (Fatty acid desaturase 2) and prevents accumulation of linolenic (18:3) containing phosphatidylcholine (PC) stimulating an increase of MGDG synthesis. Here we report that ALA10 interacts with PUB11 (plant U-box type 11), an E3 protein ubiquitin ligase, in vitro and in vivo. ALA10 is however ubiquitinated and degraded by the 26S proteasome in a PUB11-independent process. In pub11 null mutant, the proteasome-dependent degradation of ALA10 is retained and ALA10 is still subject to ubiquitination although its ubiquitination profile appears different. In the absence of PUB11, ALA10 is constrained to the ER close to chloroplasts, which is the usual location when ALA10 is overexpressed. Additionally, in this condition, the decrease of 18:3 containing PC is no longer observed. Taken together these results suggest, that ALA10 contributes in chloroplast-distal ER interacting domains, to reduce the 18:3 desaturation of PC and that PUB11 is involved in reconditioning of ALA10 from chloroplast-proximal to chloroplast-distal ER interacting domains.

Non-vesicular glycerolipids transport in plant cells

“…This association regulates ALA10 subcellular localization: in the ER close to PM when ALA10 is co-expressed with ALIS1 or in the ER close to chloroplasts when co-expressed ALIS5 (Botella et al, 2016). More recently, ALA10 localization was also shown to be impacted by its level of ubiquitination by the E3-Ubiquitin ligase PUB11, showing the complexity of the mechanisms regulating P4-ATPase localization in cells (López-Marqués et al, 2021;Salvaing et al, 2020). Overexpression of ALA10 increases the ratio MGDG/PC in leaves and the 18:2 content in chloroplastic PC, suggesting it can stimulate MGDG synthesis and enrich 18:2-PC species in chloroplasts (Botella et al, 2016).…”

Uptake and Metabolic Conversion of Exogenous Phosphatidylcholines Depending on Their Acyl Chain Structure in Arabidopsis thaliana

“…The uptake of phospholipids from soil humus could provide the roots with energy-rich acyl chains, phosphate groups, and some GPL derivatives, which participate in cell reception, signaling, and the metabolism. In green tissues, ALA10 is localized close to chloroplasts and may participate in MGDG biosynthesis, ensuring the translocation of PCs as a donor of acyl groups inside the chloroplast [ 34 , 35 , 36 ]. The expression of ALA10 involved in the uptake of GPLs from the external environment is tightly regulated along cell growth and differentiation and is regarded as the essential metabolic mechanism responsible for the acquisition of cell-specific phospholipid patterns.…”

Characteristics of Metabolites by Seed-Specific Inhibition of FAD2 in Brassica napus L

“…Fatty acid desaturase 2 (FAD2) on the endoplasmic reticulum is a key enzyme in the production of polyunsaturated fatty acids in plants. It introduces a second double bond to oleic acid (18:1) binding to phosphatidic acid choline to form linoleic acid, which is the first step in the synthesis of polyunsaturated fatty acids. , At the same time, dihydroxyacetone phosphate, the intermediate product of glycolysis, is catalyzed by glycerol 3-phosphate dehydrogenase (GPD) to form glycerol 3-phosphate, the skeleton component of triglycerides (TAGs). Then, through the Kennedy pathway, acyl coenzyme A synthesizes triacylglycerol via the catalysis of 3-phosphoglyceryl acyltransferase (GPAT), hemolytic phosphatidic acid acyltransferase (LPAT), phosphatidic acid phosphohydrolase (PAP), and diacylglycerol transferase (DGAT) on the endoplasmic reticulum. , …”