metabolic-labeling-membrane-fractionation-for-comparative-proteomic

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of <em>Arabidopsis thaliana</em> Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Max Plank Institute of Molecular Plant Physiology

Plasma membranes are dynamic compartments with key functions in solute transport, cell shape, and communication between cells and the environment. In mammalian cells and yeast, the plasma membrane has been shown to be compartmented into so-called lipid rafts, which are defined by their resistance to treatment with non-ionic detergents. In plants, the existence of lipid rafts has been postulated, but the precise composition of this membrane compartment is still under debate. Here we were able to experimentally clearly distinguish (i) true sterol-dependent "raft proteins" and (ii) sterol-independent "non-raft" proteins and co-purifying "contaminants" in plant detergent-resistant membranes. We used quantitative proteomics techniques involving (15)N metabolic labeling and specific disruption of sterol-rich membrane domains by methyl-beta-cyclodextrin. Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins. A large fraction of these proteins has functions in cell wall anchoring. We were able to distinguish constant and variable components of plant sterol-rich membrane microdomains based on their responsiveness to the drug methyl-beta-cyclodextrin. Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins. This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-beta-cyclodextrin and quantitative proteomics.

Metabolic labeling of plant tissues with (15)N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on (15)N/(14)N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio-dependent SD is used to define p-values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.

Ratio-dependent significance thresholds in reciprocal 15N-labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment.

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.

PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.

Compartimentation de la membrane plasmique r&eacute;glemente spatio-temporelle immunitaire cellulaire autonome de signalisation dans les cellules animales. Afin d&#39;&eacute;lucider la dynamique des prot&eacute;ines d&eacute;but imm&eacute;diat &agrave; la membrane plasmique de plante en r&eacute;ponse &agrave; la flagelline bact&eacute;rienne motif mol&eacute;culaire micro-organismes pathog&egrave;nes associ&eacute;s (PAMP) (flg22), nous avons utilis&eacute; l&#39;analyse par spectrom&eacute;trie de masse quantitative sur membranes r&eacute;sistants aux d&eacute;tergents (DRMs) d&#39;Arabidopsis thaliana suspension cellulaire. Cette approche a r&eacute;v&eacute;l&eacute; des changements rapides et profonds dans la composition en prot&eacute;ines DRM apr&egrave;s un traitement de PAMP, une place importante affectant proton ATPases et kinases r&eacute;cepteurs semblables, y compris le r&eacute;cepteur de la flagelline FLS2. Nous avons utilis&eacute; la g&eacute;n&eacute;tique inverse d&#39;aborder une contribution potentielle d&#39;un sous-ensemble de ces prot&eacute;ines dans les r&eacute;ponses cellulaires d&eacute;clench&eacute;s par flg22. Mutants de trois candidats (DET3, d&#39;AHA1, FER) montrent un d&eacute;faut visible dans l&#39;accumulation de PAMP-d&eacute;clench&eacute; des esp&egrave;ces r&eacute;actives de l&#39;oxyg&egrave;ne. En outre, ces mutants ont montr&eacute; alt&eacute;r&eacute;e prot&eacute;ine mitog&egrave;ne-activation de la kinase (MAPK), un d&eacute;faut de PAMP-d&eacute;clench&eacute; la fermeture des stomates, mais aussi des ph&eacute;notypes de l&#39;infection bact&eacute;rienne modifi&eacute;e, qui a r&eacute;v&eacute;l&eacute; trois nouveaux joueurs dans le contr&ocirc;le d&eacute;pendant &eacute;liciteur burst oxydatif et de l&#39;immunit&eacute; inn&eacute;e. Nos r&eacute;sultats prouvent pour des changements dynamiques dans le cloisonnement de la membrane des composants signalisation PAMP induite par l&#39;&eacute;liciteur.

PAMP (motif mol&eacute;culaire micro-organismes pathog&egrave;nes associ&eacute;s)-changements induits dans la membrane plasmique compartimentation r&eacute;v&egrave;lent de nouvelles composantes de l&#39;immunit&eacute; de la plante.

Membrana plasmatica compartimentazione regola spatiotemporally immunitario autonomo cell signaling in cellule animali. Per delucidare immediato inizio dinamiche proteine alla membrana plasmatica vegetale in risposta alla flagellina batterica pathogen-associated molecular pattern (PAMP) (flg22) abbiamo impiegato analisi quantitativa spettrometria di massa su membrane resistenti alle detergente (DRM) di cellule sospensione di Arabidopsis thaliana. Questo approccio ha rivelato rapidi e profondi cambiamenti nella composizione proteica DRM in seguito a trattamento PAMP, prominente che interessano ATPasi protonica e receptor-like chinasi, tra cui il recettore flagellina FLS2. Abbiamo impiegato la genetica inversa per affrontare un potenziale contributo di un sottoinsieme di queste proteine nelle risposte cellulari flg22-innescato. Mutanti di tre candidati (DET3, AHA1, FER) espone un cospicuo difetto nell&#39;accumulo di specie reattive dell&#39;ossigeno attivato PAMP. Inoltre, questi mutanti hanno mostrato alterata della proteina mitogene-attivata della chinasi (MAPK) attivazione, un difetto nella chiusura stomatica PAMP innescato come pure fenotipi alterati infezione batterica, che ha rivelato tre giocatori romanzo nel controllo burst ossidativo elicitori-dipendente e l&#39;immunit&agrave; innata. I nostri dati forniscono la prova per alterazioni indotte dalla elicitori dinamiche di componenti segnalazione PAMP godimento membrana.

PAMP (pathogen-associated molecular pattern)-indotti cambiamenti nella membrana plasmatica compartimentazione rivelano nuovi componenti dell&#39;immunit&agrave; di pianta.

In the growing field of plant systems biology, there is an undisputed need for methods allowing accurate quantitation of proteins and metabolites. As autotrophic organisms, plants can easily metabolize different nitrogen isotopes, resulting in proteins and metabolites with distinct molecular mass that can be separated on a mass spectrometer. In comparative quantitative experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches have been developed for metabolite identification. Here, we present an overview of currently used experimental strategies in heavy nitrogen labeling in plants and provide background on the history and function of this quantitation technique.

The use of heavy nitrogen in quantitative proteomics experiments in plants.

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Sylwia Kierszniowska

Signaling Proteomics

Max Plank Institute of Molecular Plant Physiology

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures