Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

Abstract

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.

A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis

“…Membrane Localization of AtNCL in Planta-AtNCL was reported to be localized to the vacuole by prediction and proteomics strategy (62)(63)(64)(65). In this study, p35S::AtNCL-YFP was constructed and transformed into Arabidopsis, the signal in the AtNCL-YFP transgenic seedlings was stronger near the plasma membrane after plasmolysis (Fig.…”

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

“…Accordingly, it appears that, as for CA1, CA2 has retained its ancestral C 3 role in C 4 plants. Interestingly, cytosolic b-CAs have been found associated with the plasma membranes of several plant species (Utsunomiya and Muto, 1993;Santoni et al, 1998;Kawamura and Uemura, 2003;Alexandersson et al, 2004;Mongrand et al, 2004;Benschop et al, 2007;Mitra et al, 2007), and AtbCA4 specifically localizes to Arabidopsis cell membranes (Fabre et al, 2007). Mongrand et al (2004) showed that tobacco plasma membrane lipid rafts were enriched in a cytosolic b-CA and an aquaporin, NtAQP1, which has CO 2 transport activity (Uehlein et al, 2003) and decreases the resistance of the envelope to CO 2 (Uehlein et al, 2008).…”

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

“…Unfortunately, neither REF4 nor RFR1 has been identified in any of the proteomic studies performed to date (e.g., Alexandersson et al 2004;Marmagne et al 2004;Dunkley et al 2006;Morel et al 2006;Hartman et al 2007;Heazlewood et al 2007;Lanquar et al 2007;Mitra et al 2007). The Aramemnon database for plant membrane spanning proteins (Schwacke et al 2003) annotates REF4 and its homologs as having between 10 to 12 transmembrane regions (TMRs), although for any individual protein, less than half of these score above a cutoff score of 0.5.…”