Enrichment and Identification of Integral Membrane Proteins from Barley Aleurone Layers by Reversed-Phase Chromatography, SDS-PAGE, and LC−MS/MS

Abstract

The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance; thus, little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Because detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation, and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination using a proteomics approach.

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

“…AHA1 and AHA2 are virtually expressed in all tissues and organs and function as housekeeping gene (Gaxiola et al, 2007) while other PM-H + -ATPase isoforms show some tissue specificity of expression (Morsomme & Boutry, 2000;Palmgren, 2001;Gaxiola et al, 2007). Tissue-specific localization of PM-H + -ATPase is summarised in (Hynek et al, 2006).…”

“…The AHA12 isoforms carries two large deletions and is possibly a pseudogene (Palmgren, 2001). AHA1 and AHA2 are virtually expressed in all tissues and organs and function as housekeeping gene (Gaxiola et al, 2007) (Hynek et al, 2006). …”

The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase

“…Aleurone layers were prepared from embryoless half-grains (Hynek et al, 2006) of barley 'Himalaya' (purchased from Washington State University). Isolated aleurone layers (100 mg fresh weight) were incubated in 2 mL of 20 mM sodium succinate (pH 4.2), containing 20 mM CaCl 2 , 50 mg/mL ampicillin, and 5 mg/mL nystatin.…”

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

“…These were used as the starting point from which replicate samples were prepared. Aleurone layers were isolated as described previously (Hynek et al, 2006). For each treatment, 100-mg aleurone layers were incubated in 2 mL of a baseline culture medium (20 mM sodium succinate and 20 mM CaCl 2 , pH 4.2) and incubated at room temperature with continuous gentle shaking for 24 h. When 5 mM GA 3 was added to the baseline culture, samples are referred as to GA 3 -induced aleurone layers.…”