Characterization of Arabidopsis Post-Glycosylphosphatidylinositol Attachment to Proteins Phospholipase 3 Like Genes.

Cesar Bernat-Silvestre,Yingxuan Ma,María Jesús Marcote,Kim Johnson,Fernando Aniento,Alejandro Ferrando

doi:10.3389/fpls.2022.817915

Abstract

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.

Highlights

GPI-anchored proteins (GPI-APs) are involved in diverse and crucial biological processes, including growth, morphogenesis, reproduction, and disease pathogenesis (Cheung et al, 2014)
We found that both the N-terminal as well as the C-terminal PGAP3B constructs restored MgCl2 and high temperature tolerance of per1 cells to wild-type levels, as it was the case for GFP-Per1p (Figure 2)
The per1 yeast cells were shown to have a mild phenotype in the presence of 1 M NaCl that could be restored by PGAP3B but not by PGAP3A constructs (Supplementary Figure 3C)

Summary

Introduction

GPI-anchored proteins (GPI-APs) are involved in diverse and crucial biological processes, including growth, morphogenesis, reproduction, and disease pathogenesis (Cheung et al, 2014). Once the GPI anchor is transferred onto the protein at the ER, the glycan core and the lipid moiety need to be remodeled to the mature form of the GPI anchor which is present in the GPI-APs located at the plasma membrane. The PI form of mature yeast GPI anchors contains either diacylglycerol (DAG) with a very long chain saturated fatty acid (C26:0) at the sn-2 position or ceramide containing phytosphingosine with a very long chain (C26:0) fatty acid (Kinoshita and Fujita, 2016). The major form of mammalian mature GPI anchors has 1-alkyl-2-acyl PI bearing a sn2-linked saturated fatty acid (usually stearic acid) (Kinoshita and Fujita, 2016). Only a single GPI anchor structure has been resolved, the one of PcAGP1, isolated from Pyrus communis (pear) cell suspension culture. A ceramide was detected as the lipid component of the GPI anchor of an arabinogalactan protein (AGP) isolated from Rosa sp. cell suspension culture (Svetek et al, 1999)

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Conclusion