A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

Nelson, Clark J.; Hegeman, Adrian D.; Harms, Amy C.; Sussman, Michael R.

doi:10.1074/mcp.m500414-mcp200

Cited by 92 publications

(86 citation statements)

References 58 publications

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“…58 Table 5 shows that either six or seven LRR RLKs were identified by our methanol approach using the total microsomal fraction (depending on the origin of a peptide with an exact sequence match to two LRR RLKs), whereas only two LRR RLKs were found by the Brij-58 method. At3g02880 was identified by both methods and was also found in four other proteomic analyses of the Arabidopsis plasma membrane, [25][26][27][28] suggesting it is either an abundant LRR RLK or an easily solubilized membrane protein. At3g28450 and At4g08850 were also identified in three of these proteomic studies (with the exception of Marmagne et al 25 ), whereas both At1g06840 and At5g16590 were identified by Nelson et al 28 and At5g16590 was detected by Nuhse et al 26 Our methods uniquely identified At1g62950, At5g25930, and possibly At1g35710.…”

Section: Mitra Et Almentioning

confidence: 99%

“…At3g02880 was identified by both methods and was also found in four other proteomic analyses of the Arabidopsis plasma membrane, [25][26][27][28] suggesting it is either an abundant LRR RLK or an easily solubilized membrane protein. At3g28450 and At4g08850 were also identified in three of these proteomic studies (with the exception of Marmagne et al 25 ), whereas both At1g06840 and At5g16590 were identified by Nelson et al 28 and At5g16590 was detected by Nuhse et al 26 Our methods uniquely identified At1g62950, At5g25930, and possibly At1g35710. It has been reported that a number of these identified LRR RLKs reside in lipid rafts and are enriched in Triton X-100 insoluble plasma membrane microdomains.…”

Section: Mitra Et Almentioning

confidence: 99%

“…9 Thus, it would be expected that using purified plasma membranes for proteomic analysis instead of total microsomal fractions would reduce sample complexity and increase the number of LRR RLKs identified. Interestingly, the number of LRR RLKs identified from our total membrane fractions was approximately equal to that of Alexandersson et al 27 using purified plasma membranes, although it was significantly less than the number identified by Nelson et al 28 To determine if the methanolfacilitated solubilization was compatible with phase partitioning, plasma membrane isolated from the microsomal fraction was subjected to analysis. It was determined that the number of LRR RLKs identified in a single experiment approximately doubled compared to the total microsomal fraction protocol (Table 6).…”

Section: Mitra Et Almentioning

confidence: 99%

“…The purified membrane fractions were subjected to gradient SDS-PAGE, in-gel tryptic digestion, and LC-MS/MS analysis to identify 238 putative plasma membrane proteins, 7 of which were LRR RLKs. Nelson et al 28 used whole Arabidopsis seedlings grown in shaking liquid culture to isolate 31 RLKs from purified plasma membranes, 21 of which were annotated as LRR RLKs. Here we report two complementary approaches to effectively extract and solubilize membrane proteins from total microsomal fractions isolated from whole plant tissue.…”

Section: Introductionmentioning

confidence: 99%

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Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

et al. 2007

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This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.

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Section: Mitra Et Almentioning

confidence: 99%

Section: Mitra Et Almentioning

confidence: 99%

Section: Mitra Et Almentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

et al. 2007

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“…Proteins significantly enriched in DRMs after flg22 treatment are indicated in bold (p Ͻ 0.05). Functional category (FC); Arabidopsis Genome Initiative code (AGI code); average fold-change (av fold); number of TM domains based on the consensus predicted by ARAMEMNON (TM, (17)); experimental evidence for PM association (PM, (17)(18)(19)(20)); transcriptionally co-expressed with FLS2 (46), number indicates rank of co-expressed gene according to ATTED (ATTED); elevated transcript levels in response to flg22 treatment (flg22 up, (14,21)); phosphorylated after flg22 treatment (P flg22, (15,22)); (putative) mutants of according genes were analyzed for flg22 responsiveness in this study (MA); flg22-induced ROS (ROS). enriched (enr.…”

Section: Table 1 Responding Proteinsmentioning

confidence: 99%

PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity

Keinath

Kierszniowska

Lorek

et al. 2010

Journal of Biological Chemistry

270

252

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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.To cope with the great number of potential pathogens, plants evolved specialized pattern recognition receptors (PRRs) 5 through which they detect pathogen-associated molecular patterns (PAMPs) at the cell surface (1). Within seconds to minutes after PAMP perception manifold intracellular responses occur, including ion fluxes across the plasma membrane (PM), increase of cytosolic Ca 2ϩ levels, production of reactive oxygen species (ROS) and protein phosphorylation. At later time points profound transcriptional changes, stomatal closure as well as local cell wall reinforcement take place (2).The best characterized plant PAMP perception system is the recognition of bacterial flagellin and its elicitor-active epitope, flg22, by the Arabidopsis PRR FLS2 (flagellin sensitive 2; (2)). FLS2 undergoes flg22-induced complex formation with BRl1-associated receptor kinase 1 (BAK1), which precedes and is required for FLS2 endocytosis (2, 3). Indeed, ligand-induced reduction in lateral membrane mobility of FLS2 has been observed in protoplasts (4), which could be explained by either ligand-dependent interactions of FLS2 with e.g. BAK1, the confinement of FLS2 to less mobile membrane compartments, or a combination of both. To ensure adequate perception of PAMPs and tightly regulated downstream signaling, the PM must be spatially highly organized and dynamic. In this context, the recruitment of FLS2 to specialized membrane domains seems crucial to enable ligand-induced endocytosis (5).During the past years, lateral compartmentalization has become a well-recognized topic in plant membrane research (6). The membrane raft hypothesis provides a plausible explanation for the spatial and temporal organization of biological membranes based on t...

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An Overview of the Arabidopsis Proteome

Bourguignon

Jaquinod

2008

Plant Proteomics

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A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

Cited by 92 publications

References 58 publications

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity

An Overview of the Arabidopsis Proteome

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