Dataset Information

Shot-Gun Proteomic Analysis on Roots of Arabidopsis pld?1 Mutants Suggesting the Involvement of PLD?1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis.

ABSTRACT: Phospholipase D?1 (PLD?1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLD?1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLD?1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pld?1 mutants compared to the wild type. Interestingly, PLD?1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLD?1 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 pld?1 mutants. Our results suggest a broader biological involvement of PLD?1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

SUBMITTER: Takac T

PROVIDER: S-EPMC6337374 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Shot-Gun Proteomic Analysis on Roots of Arabidopsis <i>pldα1</i> Mutants Suggesting the Involvement of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis.

Takáč Tomáš T Šamajová Olga O Vadovič Pavol P Pechan Tibor T Šamaj Jozef J

International journal of molecular sciences 20181226 1

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 shot-gun differential proteomic analysis on roots of two Arabidopsis <i>pldα1</i> mutants compared to the wild type. Interestingly, PLDα1 defi ...[more]

PMID: 30587782

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Project description:BackgroundMultidimensional protein identification technology (MudPIT)-based shot-gun proteomics has been proven to be an effective platform for functional proteomics. In particular, the various sample preparation methods and bioinformatics tools can be integrated to improve the proteomics platform for applications like target organelle proteomics. We have recently integrated a rapid sample preparation method and bioinformatics classification system for comparative analysis of plant responses to two plant hormones, zeatin and brassinosteroid (BR). These hormones belong to two distinct classes of plant growth regulators, yet both can promote cell elongation and growth. An understanding of the differences and the cross-talk between the two types of hormone responses will allow us to better understand the molecular mechanisms and to identify new candidate genes for plant engineering.ResultsAs compared to traditional organelle proteomics, the organelle-enrichment method both simplifies the sample preparation and increases the number of proteins identified in the targeted organelle as well as the entire sample. Both zeatin and BR induce dramatic changes in signaling and metabolism. Their shared-regulated protein components indicate that both hormones may down-regulate some key components in auxin responses. However, they have shown distinct induction and suppression of metabolic pathways in mitochondria and chloroplast. For zeatin, the metabolic pathways in sucrose and starch biosynthesis and utilization were significantly changed, yet the lipid biosynthesis remained unchanged. For BR, lipid biosynthesis and ?-oxidation were both down-regulated, yet the changes in sucrose and starch metabolism were minor.ConclusionsWe present a rapid sample preparation method and bioinformatics classification for effective proteomics analysis of plant hormone responses. The study highlighted the largely differing response to zeatin and brassinosteroid by the metabolic pathways in chloroplast and mitochondria.

Project description:The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Dataset Information

Shot-Gun Proteomic Analysis on Roots of Arabidopsis pld?1 Mutants Suggesting the Involvement of PLD?1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis.

Publications

Shot-Gun Proteomic Analysis on Roots of Arabidopsis <i>pldα1</i> Mutants Suggesting the Involvement of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis.

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