Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting New Roles of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis (original) (raw)
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International Journal of Molecular Sciences, 2018
Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shotgun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.
THE PLANT CELL ONLINE, 2005
PHOSPHATE TRANSPORTER1 (PHT1) genes encode phosphate (Pi) transporters that play a fundamental role in Pi acquisition and remobilization in plants. Mutation of the Arabidopsis thaliana PHOSPHATE TRANSPORTER TRAFFIC FACILITA-TOR1 (PHF1) impairs Pi transport, resulting in the constitutive expression of many Pi starvation-induced genes, increased arsenate resistance, and reduced Pi accumulation. PHF1 expression was detected in all tissues, particularly in roots, flowers, and senescing leaves, and was induced by Pi starvation, thus mimicking the expression patterns of the whole PHT1 gene family. PHF1 was localized in endoplasmic reticulum (ER), and mutation of PHF1 resulted in ER retention and reduced accumulation of the plasma membrane PHT1;1 transporter. By contrast, the PIP2A plasma membrane protein was not mislocalized, and the secretion of Pi starvation-induced RNases was not affected in the mutant. PHF1 encodes a plantspecific protein structurally related to the SEC12 proteins of the early secretory pathway. However, PHF1 lacks most of the conserved residues in SEC12 proteins essential as guanine nucleotide exchange factors. Although it functions in early secretory trafficking, PHF1 likely evolved a novel mechanism accompanying functional specialization on Pi transporters. The identification of PHF1 reveals that plants are also endowed with accessory proteins specific for selected plasma membrane proteins, allowing their exit from the ER, and that these ER exit cofactors may have a phylum-specific origin.
The Plant Journal, 2011
In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.
Plant and Cell Physiology, 2004
In order to identify integral proteins and peripheral proteins associated with the plasma membrane, highly purified Arabidopsis plasma membranes from green tissue (leaves and petioles) were analyzed by mass spectrometry. Plasma membranes were isolated by aqueous two-phase partitioning, which yields plasma membrane vesicles with a cytoplasmic-side-in orientation and with a purity of 95%. These vesicles were turned inside-out by treatment with Brij 58 to remove soluble contaminating proteins enclosed in the vesicles and to remove loosely bound contaminating proteins. In total, 238 putative plasma membrane proteins were identified, of which 114 are predicted to have transmembrane domains or to be glycosyl phosphatidylinositol anchored. About two-thirds of the identified integral proteins have not previously been shown to be plasma membrane proteins. Of the 238 identified proteins, 76% could be classified according to function. Major classes are proteins involved in transport (17%), signal transduction (16%), membrane trafficking (9%) and stress responses (9%). Almost a quarter of the proteins identified in the present study are functionally unclassified and more than half of these are predicted to be integral.
Frontiers in Plant Science, 2019
Phospholipase D alpha 1 (PLDα1) is a phospholipid hydrolyzing enzyme playing multiple regulatory roles in stress responses of plants. Its signaling activity is mediated by phosphatidic acid (PA) production, capacity to bind, and modulate G-protein complexes or by interaction with other proteins. This work presents a quantitative proteomic analysis of two T-DNA insertion pldα1 mutants of Arabidopsis thaliana. Remarkably, PLDα1 knockouts caused differential regulation of many proteins forming protein complexes, while PLDα1 might be required for their stability. Almost one third of differentially abundant proteins (DAPs) in pldα1 mutants are implicated in metabolism and RNA binding. Latter functional class comprises proteins involved in translation, RNA editing, processing, stability, and decay. Many of these proteins, including those regulating chloroplast protein import and protein folding, share common functions in chloroplast biogenesis and leaf variegation. Consistently, pldα1 mutants showed altered level of TIC40 (a major regulator of protein import into chloroplast), differential accumulation of photosynthetic protein complexes and changed chloroplast sizes as revealed by immunoblotting, blue-native electrophoresis, and microscopic analyses, respectively. Our proteomic analysis also revealed that genetic depletion of PLDα1 also affected proteins involved in cell wall architecture, redox homeostasis, and abscisic acid signaling. Taking together, PLDα1 appears as a protein integrating cytosolic and plastidic protein translations, plastid protein degradation, and protein import into chloroplast in order to regulate chloroplast biogenesis in Arabidopsis.
Mutation of the TGD1 Chloroplast Envelope Protein Affects Phosphatidate Metabolism in Arabidopsis
THE PLANT CELL ONLINE, 2005
Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.
Plant physiology, 2016
The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty...
THE PLANT CELL ONLINE, 2008
The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-toplastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid. Extraplastidic TGD4 Protein Requires the Arabidopsis Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in This information is current as of June 15, 2015 Supplemental Data http://www.plantcell.org/content/suppl/2008/08/01/tpc.108.061176.DC1.html References
THE PLANT CELL ONLINE, 2008
Vesicle budding in eukaryotes depends on the activity of lipid translocases (P 4 -ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P 4 -ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a b-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.