Enrichment and Identification of Integral Membrane Proteins from Barley Aleurone Layers by Reversed-Phase Chromatography, SDS-PAGE, and LC-MS/MS Radovan Hynek,† Birte Svensson,† Ole Nørregaard Jensen,‡ Vibeke Barkholt,† and Christine Finnie*,† Enzyme and Protein Chemistry Group, BioCentrum-DTU, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark, and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark Received June 12, 2006
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 SDSPAGE 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. Keywords: batch reversed-phase chromatography • cereals • H+-ATPase • isoforms • seed proteins
Introduction The germination process in cereals is of particular importance to the malting, feed, and food industries. Much effort has been put into understanding the mechanisms of cereal seed development and germination at the molecular level. The cereal aleurone layer plays a pivotal role in germination by synthesizing and secreting enzymes that break down reserves stored in the starchy endosperm.1 The gibberellic acid signal produced by the embryo upon seed imbibition is perceived at the plasma membrane of the aleurone layer,2,3 and a signaling cascade is initiated that results in synthesis and secretion of hydrolytic enzymes and culminates in controlled death of the aleurone cells.4 In addition to its key role in germination, the barley aleurone layer represents a unique system for the study of plant signaling because (i) it is easily separated from other seed tissues, (ii) it shows specific, well defined responses to the plant hormones gibberellic acid and abscisic acid, and (iii) the dissected aleurone layers can be maintained and manipulated in culture. Proteomics techniques have recently been applied to barley seeds to identify proteins involved in seed develop* To whom correspondence should be addressed. Christine Finnie, Enzyme and Protein Chemistry Group, BioCentrum-DTU, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; E-mail:
[email protected]; Tel: +45 45 25 27 39; Fax: +45 45 88 63 07. † Technical University of Denmark. ‡ University of Southern Denmark. 10.1021/pr0602850 CCC: $33.50
2006 American Chemical Society
ment and germination.5-8 The proteomes of dissected embryo, aleurone layers, and endosperm were furthermore analyzed individually.9 The above studies were limited to soluble proteins. However, about a third of all proteins encoded by genomes are estimated to be membrane proteins. Because many of the events of seed germination involve membranes, an analysis of the membrane proteins is clearly necessary for an in-depth understanding of the germination process in which membrane proteins of the aleurone layer are of particular interest. Membrane proteins, however, are difficult to analyze due to their hydrophobicity and low abundance and detailed insight into the membrane proteins involved in seed germination is still lacking. The hydrophobicity of intrinsic membrane proteins promotes their precipitation at their isoelectric points, making classical twodimensional gel electrophoresis highly problematical. Pioneering 2-DE studies of Arabidopsis plasma membranes10,11 resulted mainly in the identification of peripheral membrane proteins. However, the combination of one-dimensional SDS-PAGE with LC-MS/MS has proved to be a useful strategy for separation and identification of integral membrane proteins.12-14 Plasma membranes with relatively low contamination by endomembranes can be prepared by aqueous two-phase partitioning.15 During homogenization, soluble proteins become entrapped in membrane vesicles or bind to membranes via nonspecific ionic interactions. Removal of such soluble Journal of Proteome Research 2006, 5, 3105-3113
3105
Published on Web 10/04/2006
research articles contaminants is normally carried out using elevated ionic strength in the presence of mild detergents, e.g., Brij-58, which inverts the membrane vesicles, or Triton X-100.16,17 However, soluble proteins were still identified in Arabidopsis plasma membrane fractions even after washing.12,13 Several aspects of protein identification using mass spectrometry are problematic for hydrophobic proteins; (i) longer hydrophobic sequence stretches are not cleaved by trypsin, (ii) strongly hydrophobic peptides are difficult to extract from the gel after digestion, and (iii) hydrophobic peptides remain bound to the reversed-phase microcolumns routinely used for sample cleanup and to reversed-phase columns coupled with MS/MS systems. As a result, only a few peptides are normally identified for each membrane protein. Thus, the presence of even low amounts of soluble proteins can hamper successful identification of intrinsic membrane proteins. In the past few years, proteome analyses have been reported for various plant endomembranes18 including the chloroplast envelope, thylakoid membrane, mitochondrion, tonoplast, peroxisome and glyoxysome, nuclear envelope, endoplasmic reticulum, and Golgi apparatus. Proteomic identification of integral plasma membrane proteins has so far primarily been reported for Arabidopsis,12,13 although 2-DE based analyses, mainly identifying peripheral proteins, have been carried out with rice plasma membranes.19 Most of the above studies were based on membranes isolated from green tissues or from cell cultures. Isolation of plasma membranes from seed aleurone layers is difficult due to the scarcity of the tissue and the presence of secondary metabolites that interfere with protein extraction. Previously, the isolation of plasma membranes from aleurone layers using two-phase partitioning was reported20,21 but the protein profiles of such membranes have not been analyzed in any detail. In the present study, a plasma membrane-enriched fraction was prepared from aleurone layers and carefully characterized by western blotting for marker proteins, both for the plasma membrane and the presence of contaminating endomembranes and soluble proteins. Batch reversed-phase chromatography was introduced to reduce the amount of soluble contaminants and served also to fractionate the membrane proteins. This new procedure, enriched for hydrophobic proteins, led to the first identification of integral membrane proteins from aleurone layers and will facilitate proteome analysis of the aleurone layer membranes during germination.
Experimental Methods Barley Aleurone Layer Preparation. Seeds of the hull-less barley cultivar Himalaya (1998 harvest) were purchased from Washington State University, Pullman, WA, U.S.A. The embryocontaining part and the distal 1-2 mm were cut off with a sharp scalpel, and the embryoless half-seeds were surface sterilized in 70% ethanol for 1 min followed by 5 washes in distilled water. Half-seeds were incubated in batches of 50 for 4 days at 4 °C in 25 mL water containing 50 µg/mL ampicillin and 5 µg/mL nystatin. The starchy endosperm was thereafter carefully scraped away from the aleurone layers during multiple water washes at room temperature until the wash water was clear. Cleaned aleurone layers were frozen in liquid nitrogen and stored at -80 °C. Aleurone Plasma Membrane Preparation by Two-Phase Partitioning. Plasma membranes were obtained by aqueous two-phase partitioning essentially as described.15 The twophase system was initially established using 50 g spinach leaves, 3106
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which are readily available and allow visual inspection of chlorophyll partitioning. This procedure was scaled down for 5-10 g of spinach leaves and finally adapted for barley aleurone layers as follows: 10 g aleurone layers (fresh weight) were ground to powder in liquid nitrogen and homogenized in a ground glass homogenizer on ice with 40 mL of homogenization buffer (50 mM MOPS, 5 mM EDTA, 0.33 M sucrose, pH 7.5), containing 5 mM DTT, 5 mM ascorbate, 0.6% (w/v) poly(vinylpyrrolidone) and protease inhibitors (EDTA-free complete mini protease inhibitor cocktail, according to manufacturers recommendations, Roche Diagnostics, Germany). The homogenate was filtered through 4 layers of cheesecloth, protease inhibitors were added once more followed by centrifugation at 10 000 g (9200 rpm, SS-34 rotor, JA-14 Beckmann) for 15 min at 4 °C to remove debris. The supernatant was ultracentrifuged at 55 000 g (27 000 rpm, 70 Ti rotor, Beckmann) for 35 min at 4 °C to pellet the microsomal membranes. The supernatant (cytosolic fraction) was decanted and the microsomal pellet, containing 7.3 mg protein, was resuspended in 6 mL of resuspension buffer (5 mM potassium phosphate, pH 7.8, 0.33 M sucrose, 3 mM KCl, 0.1 mM EDTA) containing 1 mM DTT. A plasma membrane-enriched fraction was obtained by aqueous two-phase partitioning20 of the microsomal fraction in a system of the following composition: 6.2% w/w PEG 3350 and 6.2% (w/w) dextran T500, 5 mM potassium-phosphate pH 7.8, 3 mM KCl, 11.3% (w/w) sucrose, final weight 24 g. Phase separation was achieved by centrifuging at 1000 g in a swingingbucket rotor at 4 °C for 5 min. The upper and lower phases were successively re-extracted three times as described.15 The final upper phases, enriched for plasma membranes, were combined, diluted 1:1 with resuspension buffer, and ultracentrifuged (90 min, 110 000 g, 4 °C) for recovery of plasma membranes. A soft paintbrush was used to resuspend the pellet in 200 µL of storage buffer (25 mM MOPS-KOH, pH 7.5, 0.25 M sucrose, 0.5 mM EDTA) containing 1 mM DTT and protease inhibitor cocktail. The final lower phases (depleted for plasma membrane) were combined, diluted 10 times with resuspension buffer, and ultracentrifuged as above to recover membranes. The total protein content of the upper and lower phase fractions was determined using the Popov assay22 and BSA as a reference, to approximately 0.3 and 2.5 mg protein, respectively. Removal of Soluble Proteins from the Membrane Fraction using Batch Reversed-Phase Chromatography. Membrane proteins were delipidated prior to reversed-phase chromatography. Membranes (50 µL) were mixed well with 50 µL of chloroform, 200 µL of methanol, and 500 µL of water and centrifuged briefly. Proteins formed an interface between the chloroform and methanol phases. The top phase was discarded, 250 µL of methanol was added, mixed, and centrifuged to pellet the precipitated proteins. The proteins were dissolved in solubilizing buffer (50 mM MES, 50 mM Tris, 0.5% (w/v) SDS, 1 mM EDTA, pH 7.3). Batch reversed-phase chromatography was performed in Eppendorf tubes at room temperature. Nucleosil 300-5 C4 (Macherey-Nagel, Durey, Germany) was used as stationary phase, and mobile phases were prepared with increasing concentrations of 2-propanol (Sigma) in 0.15% aq. TFA (Fluka). Dry Nucleosil C4 (100 µL) was wetted with 100% 2-propanol (500 µL) and equilibrated with 0.15% aq. TFA. Delipidated membrane proteins (approximately 140 µg in 150 µL of solubilizing buffer) were added to the pretreated Nucleosil C4 and mixed well, followed by addition of 0.15% aq. TFA (400 µL), mixing, and centrifugation. Stepwise elution was done by
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Integral Membrane Proteins from Barley Aleurone Layers
adding a given eluent in two portions of 200 µL followed by centrifugation to sediment the stationary phase. Eluates were vacuum-dried. Elution is highly sensitive to small differences in 2-propanol concentration. Practically, this was controlled by preparing an eluent series of 2-propanol concentrations and determining those leading to elution of the desired proteins. The chosen eluents were stored in aliquots at -18 °C for subsequent use to ensure reproducibility of elution profiles. SDS-PAGE and Western Blotting. Membrane proteins were separated on 4-12% BisTris NuPAGE gels (Invitrogen) run in 50 mM MES, 50 mM Tris, 0.5% (w/v) SDS, 1 mM EDTA, pH 7.3 and were visualized by silver staining.23 Gels used for in geldigestion and mass spectrometry were stained using colloidal Coomassie Brilliant Blue G-250.24 For western blotting, approximately 3 µg of protein from each fraction was separated by SDS-PAGE as above followed by electroblotting onto nitrocellulose membranes (Hybond-N, GEHealthcare). Blots were probed with primary antibodies recognizing the N-terminus of the Arabidopsis thaliana plasma membrane H+-ATPase (kindly provided by Anja Thoe Fuglsang, Royal Veterinary and Agricultural University, Frederiksberg, Denmark), luminal binding protein BiP and tonoplast intrinsic protein (kindly provided by Maarten Chrispeels, University of California, San Diego, CA, U.S.A.), β-amylase (kindly provided by Evan Evans, University of Adelaide, Australia), and serpin (kindly provided by Jørn Hejgaard, Technical University of Denmark). Polyclonal goat antirabbit immunoglobulins conjugated with alkaline phosphatase (DakoCytomation, Denmark) were used as secondary antibody and 5-bromo-4-chloro-3indolyl phosphate/nitroblue tetrazolium (SIGMA FAST, Sigma) was used for detection. In-Gel Digestion of Proteins for Peptide Mass Mapping. Ingel digestion with trypsin was adapted from standard procedures.25 Briefly, the bands of interest were excised from the SDS-PAGE gel, cut into 1 mm3 cubes, and washed in 1:1 (v/v) 100 mM NH4HCO3 in acetonitrile. Cys residues were reduced with DTT and alkylated with iodoacetamide. Digestion was carried out with 10 µg/mL sequencing grade trypsin (Promega) in 50 mM NH4HCO3 at 37 °C overnight. Peptides were extracted from the gel in 1:1 (v/v) 25 mM NH4HCO3 in acetonitrile by sonication for 10 min. The supernatant was recovered, and 35% acetonitrile, 0.1% TFA was added to the gel particles followed by sonication for 15 min. This step was repeated with 70% acetonitrile, 0.1% TFA. All extracts were pooled and vacuum-dried. Mass Spectrometry, Protein Identification, and Data Analysis. LC-MS/MS was carried out using an LC-Packings Ultimate 3000 nanoflow system (LC Packings, Amsterdam, The Netherlands) coupled to an ESI-Q-TOF Micro tandem mass spectrometer (Waters/Micromass, Manchester, UK). Peptides were solubilized in 0.5% acetic acid and loaded with a flow rate of 3 µL/min onto a custom-made 1 cm precolumn (75 µm i.d. fused silica with kasil-frits retaining Reprosil C18, 3.5 µm reversedphase particles (Dr. Maisch GmbH, Germany)). Reversed-phase HPLC was carried out with a flow rate of 0.1 µL/min through a custom-made 8 cm analytical column (50 µm i.d. packed with Reprosil C18, 3.5 µm reversed-phase particles). Peptides were eluted directly into the ESI source using a stepped linear gradient (0-10% B, 5 min; 10-50% B, 30 min; and 50-100% B, 5 min; solvent A was 0.5% acetic acid, and B was 90% acetonitrile, 0.5% acetic acid). Mass- and charge-dependent collision energies were used for peptide fragmentation. Protein identification was performed using the Virtual Expert Mass Spectrometrist (VEMS) software V 3.026 to search the
Figure 1. Immunoblot analysis of aleurone layer protein fractions. Western blotting with antiserum against: (A) plasma membrane H+-ATPase; (B) luminal binding protein BiP; (C) tonoplast intrinsic protein TIP; (D) β-amylase. Equal amounts of protein (3 µg) were loaded from the plasma membrane enriched fraction (upper phase, U), endomembrane fraction (lower phase, L), microsomal fraction (M), and cytosolic (C) fraction.
Institute of Genome Research (TIGR) barley gene index (HvGI) release 9.0 (September 15, 2004). The following search parameters were used: one missed cleavage, carbamidomethylated cysteines (fixed modification), oxidized methionine (variable modification), peptide mass accuracy 0.5 Da, MS/MS peptide fragment mass accuracy 0.5 Da, VEMS score threshold for peptides 10, VEMS score threshold for protein 40. For a protein identification to be considered reliable, sequences of at least two independent peptides were required. Independent peptides differ in primary sequence; thus, charge variants, oxidized, or otherwise modified forms and missed cleavage forms of the same peptide are not considered to be independent. Identified TC sequences were submitted to a BLAST search for homologous protein sequences. Average hydrophobicity (GRAVY) was calculated using the SOSUI server (http://sosui.proteome.bio. tuat.ac.jp/sosui_submit.html), and transmembrane domains were predicted by TMpred (http://www.ch.embnet.org/software/ TMPRED_form.html).
Results and Discussion Isolation of Plasma Membrane Enriched Fraction from Barley Aleurone Layers. Aqueous two-phase partitioning15 was used for isolation of a plasma membrane-enriched fraction from barley aleurone layers. Western blotting of marker proteins from plasma membrane (H+-ATPase), endoplasmic reticulum (luminal binding protein BiP), and tonoplast (tonoplast intrinsic protein) was used to evaluate the two-phase partitioning. The highest enrichment for barley aleurone plasma membranes and minimum contamination by nonplasma membranes was achieved at 3 mM KCl, 6.2% (w/w) dextran T500, and 6.2% (w/w) PEG 3350 (data not shown) in agreement with previously reported conditions.20 The approximate yield of plasma membrane proteins was 30 µg/g fresh weight of aleurone layers. Western blotting for marker proteins (Figure 1) confirmed that the plasma membrane H+-ATPase was enriched more than 7-fold in the upper phase compared with the original microsomal fraction, and only a trace of the H+ATPase, corresponding to one twelfth of the band intensity in the upper phase, was detected in the lower phase. Luminal binding protein was barely detected in the upper phase but gave a strong reaction in the cytosolic and microsomal fractions and the lower phase (Figure 1). The tonoplast intrinsic protein antibody recognized bands in all fractions (Figure 1). The presence of tonoplast integral protein in the plasma membrane fraction was not unexpected because TIP had previously been identified in plasma membrane fractions from Arabidopsis.12,13 Journal of Proteome Research • Vol. 5, No. 11, 2006 3107
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During homogenization, soluble proteins become trapped in membrane vesicles, although this is sometimes overlooked in membrane protein preparations.27 The soluble protein β-amylase was used as a marker of contamination of the aleurone layer membranes. β-Amylase originates from the starchy endosperm residual in aleurone layer preparations, but because it is highly abundant, traces are normally detected in aleurone layer extracts. β-Amylase present in aleurone layer membrane preparations is thus a result of nonspecific interactions or encapsulation in membrane vesicles. Western blotting showed that β-amylase, as expected, occurred predominantly in the cytosolic fraction, but significant amounts were present also in lower and upper phases after two phase partitioning of membranes (Figure 1). Several washing procedures including treatment with high ionic strength and detergents Triton X-100 or Brij 5813,16,17 can be used to reduce the amount of soluble proteins in membrane preparations, but such treatments failed to reduce the β-amylase content of the aleurone layer plasma membrane fraction significantly (data not shown). Soluble contaminating proteins were also identified in an Arabidopsis plasma membrane fraction after washing with Brij 58 in the presence of KCl.13 These results suggested that an alternative strategy for reduction of the soluble protein content in membrane preparations would be valuable. Removal of Soluble Proteins from the Plasma Membrane Fraction by Batch Reversed-Phase Chromatography. A batchwise reversed-phase chromatographic procedure was developed to ensure reduced contamination of aleurone layer plasma membrane fractions with soluble proteins and to fractionate integral membrane proteins. The sample was applied to Nucleosil 300-5 C4 resin and washed and proteins were eluted with a step gradient of 2-propanol. The procedure was initially optimized using a mock-sample prepared from spinach leaves to which 10% (w/w) barley seeds was added during homogenization. This allowed preparation of spinach leaf membranes contaminated with β-amylase, suitable for optimization of the reversed-phase chromatography of intrinsic membrane proteins. Conditions established for this spinach model (data not shown) were applied to the barley aleurone layer plasma membrane fraction. Western blotting (Figure 2A) showed all β-amylase to be eluted at 54.25% 2-propanol. The vast majority of the plasma membrane H+-ATPase eluted at 80% 2-propanol, and no β-amylase was observed in this fraction. Another soluble protein (serpin) gave a similar elution profile as β-amylase, supporting that these conditions may be suitable for separation of intrinsic membrane proteins from soluble proteins. Silver staining (Figure 2B) showed that a significant proportion of protein was eluted with 54.25% of 2-propanol. The more hydrophobic proteins were subsequently separated in two fractions eluted at 54.5% and 80% 2-propanol. However, it cannot be ruled out that some very hydrophobic proteins may remain bound to the resin even at this concentration of 2-propanol. The reproducibility of this batch-wise reversed-phase chromatography was proven in three subsequent experiments that gave rise to the same protein profiles on SDS-PAGE (data not shown). The combination of reversed-phase chromatography with SDS-PAGE seemed effective both for removal of the majority of the soluble proteins from integral membrane proteins and for obtaining a fraction enriched in integral membrane proteins as eluted by 80% 2-propanol (Figure 2). This system could be further developed for reversed-phase HPLC with gradient elu3108
Journal of Proteome Research • Vol. 5, No. 11, 2006
Figure 2. Fractionation of proteins by reversed-phase chromatography. About 140 µg of plasma membrane protein was fractionated by reversed-phase batch chromatography and eluted by 2-propanol. (A) Western blotting of fractions eluted by 54.25% (lane 1), 54.5% (lane 2), and 80% (lane 3) 2-propanol, with antisera against plasma membrane H+-ATPase, β-amylase, and serpin. (B) Corresponding silver stained SDS-PAGE gel. (C) Proteins eluted by 80% 2-propanol were separated by SDS-PAGE and coomassie stained for subsequent in-gel digestion of proteins. Ten times more protein was applied to this gel than to the corresponding silver stained lane in B. Bands analyzed by LCMS/MS are indicated by numbers.
tion, which combined with SDS-PAGE could provide a two-dimensional technique for separation of intrinsic membrane proteins. Mass Spectrometric Identification of Barley Aleurone Layer Membrane Proteins. Proteins eluted from the reversed phase system by 80% 2-propanol were applied to SDS-PAGE. Ten bands (Figure 2C) were chosen and excised, in-gel digested with trypsin, and subjected to LC-MS/MS. Relatively few barley
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Integral Membrane Proteins from Barley Aleurone Layers Table 1. Proteins Identified in the Barley Aleurone Layer Plasma Membrane Fraction band/ kDaa
1/105
2/75 3/65
4/55
5/50 6/45
7/42
8/37
TC
homologous proteinb
TC139045
AAN15220 plasma membrane H+-ATPase (Hordeum vulgare) CAD29296 plasma membrane H+-ATPase (Oryza sativa) AAT81733 plasma membrane H+-ATPase (O. sativa) NP•568051 V-type ATPase (Arabidopsis thaliana) BAA02717 inorganic pyrophosphatase (H. vulgare) BAB18681 inorganic pyrophosphatase (H. vulgare) BAB18682 V-type ATPase (H. vulgare) P80284 protein disulfide-isomerase precursor (H. vulgare) AAO67355 endosperm-specific β-amylase 1 (H. vulgare) AAA34269 storage protein (Triticum aestivum) CAA52636 ATP synthase β-subunit (T. aestivum) 1802402A globulin 2 (Zea mays) AAT93903 unknown protein (O. sativa) AAK07827 mitochondrial processing peptidase β-subunit (Cucumis melo) CAA67492 atpA (Secale cereale) AAW34190 B3-hordein (Hordeum chilense) BAD28134 putative pseudouridine synthase 1 (O. sativa) CAA66232 protein z-type serpin (H. vulgare) CAA64599 serpin (H. vulgare) CAA39602 aspartic proteinase (H. vulgare) XP•463430 putative permease 1 (O. sativa) XP•479571 r40 g2 protein (O. sativa) CAA42901 glyceraldehyde 3-phosphate dehydrogenase (H. vulgare) AAT77030 putative steroleosin-B (O. sativa) XP•463436 aspartate aminotransferase (O. sativa) BAD33626 polyubiquitin 2 (O. sativa) AAQ74238 caleosin 1 (H. vulgare) ABA98146 11-beta-hydroxysteroid dehydrogenase, putative (O. sativa) CAA57647 voltage-dependent anion channel (VDAC) (T. aestivum) XP•467495 ATP/ADP translocator (O. sativa) AAN17454 hypersensitive-reaction induced protein 4 (H. vulgare) T06212 glucose and ribitol dehydrogenase homologue (H. vulgare) P46274 voltage-dependent anion channel (VDAC) (T. aestivum) AAN17457 hypersensitive-reaction induced protein 1 (H. vulgare) AAT40548 putative vicilin (Solanum demissum) BAD27962 unknown protein (O. sativa) CAA57994 high molecular weight oleosin (H. vulgare) CAA57646 voltage-dependent anion channel (VDAC) (T. aestivum) P22244 protein synthesis inhibitor I (H. vulgare) CAA57995 low molecular weight oleosin (H. vulgare) 1BGP barley grain peroxidase 1 (H. vulgare) ABB51093 reticulon (H. vulgare) XP•450518 putative oleosin (O. sativa) AAA62698 ubiquitin (H. vulgare) XP•467970 mitochondrial phosphate transporter (O. sativa) NP•916988 guanine nucleotide-binding protein β-subunit-like (O. sativa) NP•922119 putative β-tonoplast intrinsic protein (O. sativa)
TC143797 TC151705 TC147103 TC131606 TC131805 TC139247 TC146674 TC146664 TC139034 TC130729 TC131411 TC148443 TC147029 AJ461865 TC138764 TC134718 TC139095 TC139354 TC146792 TC132320 TC130737 TC131363 TC138361 TC131172 TC134494 TC147305 TC132308 TC147016
9/32
TC146246 TC132099 TC131559 TC146872 TC146912 TC132968 TC149025 TC139073 TC131984 TC131896 TC139227 TC130885 TC147119 TC139387 TC130753 TC131596 TC131070
10/22
TC147137
peptidesc
scored
MW kDae
GRAVYf
TMDg
14 (17)
950
105
0.112
11
6 (9) 4 7 17 (18) 12 (13) 7 (8) 3 2 11 (13) 9 (11) 10 6 3
432 203 281 701 518 379 87 88 688 694 617 274 125
105 105 94 79 80 68 56 60 72 59 50 53 59
0.088 0.096 0.042 0.618 0.617 -0.208 -0.230 -0.407 -1.093 -0.062 -0.503 0.059 -0.302
11 10 7 14 15 2 1 0 1 1 1 4 0
3 2 2 10 (11) 9 (10) 6 4 17 (24) 8 (11)
121 86 65 613 492 291 194 826 517
55 31 64 43 43 54 57 38 37
-0.107 -0.581 -0.536 -0.025 0.071 0.045 0.544 -0.511 -0.126
0 2 0 3 2 3 12 1 0
3 2 2 3 2
165 99 51 144 94
39 45 17 34 41
-0.083 -0.062 -0.447 -0.559 -0.010
2 1 0 1 1
2
53
29
-0.223
1
8 (10) 8 (9)
473 364
42 32
-0.101 -0.085
5 1
6
322
32
-0.282
1
5
278
29
-0.115
1
5
250
31
-0.151
0
4 3 (4) 3 3
251 187 205 199
60 32 18 30
-0.271 0.295 0.158 -0.191
4 6 2 0
3 2 2 2 2 (3) 2 2
147 88 123 94 81 61 53
30 16 34 23 16 18 39
-0.131 0.274 -0.256 0.287 0.358 -0.775 0.221
2 2 0 3 2 0 3
2
48
36
-0.124
0
2
105
28
0.564
6
a Band numbers refer to Figure 2C. Molecular weights according to migration on SDS-PAGE. b Most similar protein identifed by BLAST search of the identified TC sequence. c Number of independent peptides matching the TC sequence (in brackets, total number of sequenced peptides matching the TC sequence including peptide forms with different charge states, methionine oxidation levels and missed cleavages). d VEMS score.26 e Molecular weight calculated for the most homologous protein sequence. f Grand Average Hydrophathy score for the most homologous protein sequence. g Transmembrane domains predicted for the most homologous protein sequence.
sequences are present in protein sequence databases. One-third of the soluble protein identifications from the barley seed proteome6-8 were based on EST sequence information in the form of TC sequences in the TIGR barley gene index. Because membrane proteins in general are less well studied than soluble
proteins, they are likely to be under-represented in the sequence databases. Therefore, in this study, the TIGR barley gene index was used for searching with peptide masses from the membrane protein samples. In total, 46 barley TC sequences were identified in the 10 Journal of Proteome Research • Vol. 5, No. 11, 2006 3109
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Table 2. Evidence for Protein Isoforms among Identified Barley Aleurone Layer Plasma Membrane Proteins banda
1
3
8/9
9
3110
peptide sequencesb
zc
scored
LGmGTNmYPSSALLGQSK (LGmGTNMYPSSALLGQSK) (LGMGTNMYPSSALLGQSK) LGmGTNMYPSSALLGQNK (LGMGTNmYPSSALLGQNK) ADIGIAVADATDAAR ADIGIAVDDATDAAR NPGDEVFSGSTCK GPGDGVYSGSTVK IDQSGLTGESLPVTK IDQSALTGESLPATK mITGDQLAIGK (MITGDQLAIGK) AGYNELNQMAEEAK (AGYNELNQmAEEAK) ELSEIAEQAKg GAPEQIITLCNCK LGDIIPADAR LGDIVPADAR LSQQGAITK AWDNLLQNK EAQWATAQR VEIFGLNK LLEGDPLK VENQDAIDACmVGMLADPK SLGVAYQDVPDGR TAQDDFNK TDALDAAGNTTAAIGK DTSGPSLNILIK QFNTIPGLMEGTAKPDYATCVK (QFNTIPGLmEGTAKPDYATCVK) AAVIGDTIGDPLK KYIEAGNSEHAR (YIEAGNSEHAR) (YIEAGNSEHAR) FTIFNFGAQK TDALDAAGNTTAAIGK ISTDASIK IATYANAR AADVGADLVGK TGAATNVIFGLALGYK AANEIEPALKK SQPCHYSVGK MVEEVR NIPEDDPR SHSSTAVGAELSHNFPR KADLILGEIQSQIK KNEAILSDLQTQVK FTLTTCTPEGVAITAAGTR TILSFAVPDQK SILSLVVPDQR GVLSLPFPYQK GDSLTASYYHFVEK SLCTISAEVDTK ADLILGEIQSQIK LAEPGLHFFNPCAGELVAGTLSTR DSVLAFSENVPGTSSK ENILNFSHSVSGTSAK mELDSLFEQK (MELDSLFEQK) mNLDDVFEQK AmNEINAAAR AmNDINAAQR YLAGVGIAR YLSGVGIAK EQIQSYVFDVIR QAITDGLR AVLEELEK DIGDQIR
2 (2) (2) 2 (2) 2 2 2 2 2 2 2
82 (79) (47) 55 (51) 82 80 79 58 71 50 65 (53) 84 (16) 53 49 49 46 46 44 41 39 39 37 78 15 99 69 65 (50) 64 60 (59) (39) 58 44 40 39 37 36 35 29 24 16 83 70 24 62 69 54 29 62 41 36 82 79 50 65 (43) 58 48 24 37 33 28 25 25 17
protein
plasma membrane H+-ATPase (2 isoforms)
inorganic pyrophosphatase (2 isoforms)
voltage-dependent anion channel (3 isoforms)
hypersensitive-reactioninduced protein (2 isoforms)
Journal of Proteome Research • Vol. 5, No. 11, 2006
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 (3) 2 3 (2) (3) 2 2 2 2 2 2 3 3 2 2 3 2 2 2 2 2 2 2 2 2 3 2 3 2 (2) 2 2 2 2 2 2 2 2 2
contained in TCe
isoformf
TC139045
*
TC143797
**
TC143797 TC139045 TC139045 TC151705 TC139045 TC151705 TC139045, TC143797
** * * ** * **
TC143797
**
TC139045 TC139045 TC151705 TC139045 TC139045, TC151705 TC139045 TC139045 TC139045 TC139045 TC139045 TC143797 TC143797 TC131606, TC131805 TC131606, TC131805 TC131606, TC131805
*
TC131606, TC131805 TC131606
** * * * * * ** **
*
TC131606 TC131606, TC131805 TC131606, TC131805 TC131606, TC131805 TC131606, TC131805 TC131606, TC131805 TC131606 TC131805 TC131606, TC131805 TC131606, TC131805 TC131984 TC146872 TC147016 TC131984 TC146872 TC131984 TC147016 TC146872 TC146872 TC146872 TC132099 TC146912 TC132099 TC132099
*
* ** *** * ** * *** ** ** ** * ** * *
TC146912 TC146912 TC132099 TC146912 TC132099 TC146912 TC132099 TC132099 TC132099
** ** * ** * ** * * *
* **
research articles
Integral Membrane Proteins from Barley Aleurone Layers Table 2. (Continued) banda
protein
peptide sequencesb
zc
scored
9
Oleosin (3 isoforms)
TPDYVEEAR GSYVQVQHGGQYGAGQQQHGR TQQAGHAIQSR GGVYGGGSAVGPDYMR (GGVYGGGSAVGPDYmR) GVLGGGGAFADR HPPGADQLDHAK GDDDYYGGHGQGHNQPAAVTLAK
2 3 3 2 (2) 2 3 4
74 66 65 56 (47) 53 35 25
contained in TCe
isoformf
TC139073 TC139073 TC139073 TC139387
* * * **
TC139227 TC139227 TC139387
*** *** **
a Band numbers refer to Figure 2C and Table 1. b Peptides identified by MS/MS. Lower case m designates oxidized methionine. Additional forms of the same peptide are given in parentheses. Where the corresponding peptides from two isoforms have been identified, the amino acid differences are bold and italic. Missed tryptic cleavage sites are indicated in italic. c Charge state of sequenced peptide. d VEMS peptide score.26 e TC sequences containing the peptide sequences. f Peptides that can be assigned to a specific isoform are indicated; different isoforms are distinguished by the number of asterisks. g The isoform encoded by TC139045 contains an R in place of N, creating a new tryptic cleavage site; thus, the sequence begins at the subsequent E.
analyzed bands. One or more transmembrane helices were predicted for 34 (74%) of these identifications. This is a relatively high proportion compared with others,13 who used Brij 58 in 0.2 M KCl for removal of soluble proteins from an Arabidopsis plasma membrane fraction and identified 128 proteins with predicted TMDs (42%) out of 304 total identifications. This suggests that reversed-phase chromatography is a promising and efficient method to enrich for integral membrane proteins, although a systematic study should be carried out to confirm the general usefulness of this tool. However, despite the general great reduction in amount of soluble proteins achieved by the reversed-phase chromatography procedure, a few soluble proteins were still identified. These included β-amylase and serpin, which were identified even though they were not detected by western blotting (Figure 2A) and glyceraldehyde-3-phosphate dehydrogenase found also in Arabidopsis plasma membranes.13 The identification of several soluble storage proteins in the aleurone layer membrane preparation (e.g., TC139034 and TC131411 in band 4; Table 1) probably reflects their very high abundance, analogous to the usual presence of RuBisCo in membranes isolated from green tissues. These results highlight both the sensitivity of the mass spectrometric identifications and the necessity of including measures to reduce the content of soluble protein in membrane preparations. The plasma membrane H+-ATPase was identified in band 1 (Table 1) by MS/MS fragment ion fingerprinting of 22 independent peptides. Isoform-specific peptides (Table 2) showed that at least two H+-ATPase isoforms with 80% sequence identity were present. Of the 22 peptides, 11 were specific to TC139045, distinguishing this sequence from the only fulllength barley H+-ATPase sequence in the NCBI database, AAN15220 with 95% sequence identity to TC139045. Eight peptides were specific to a combination of two partial and nonoverlapping TC sequences, TC143797 and TC151705, sharing each about 95% identity with a rice sequence AAT81733. The remaining three peptides were common to both sequences. Thus, at least two new isoforms of the barley plasma membrane H+-ATPase were identified in aleurone layer plasma membranes. TC sequences homologous to two different subunits of the V-type H+-ATPase (94 kDa and 68 kDa; in bands 2 and 3), two isoforms (Table 2) of the inorganic pyrophosphatase H+-PPiase (band 3), and TIP (band 10) in agreement with the western blot (Figure 1) were identified. These three proteins, although originally assumed to be exclusive to the tonoplast, have been immunogold-localized to the plasma membrane of pea seeds.28
H+-PPiase has also been immunogold-localized and H+-PPiase activity detected in highly purified plasma membranes from cauliflower.29 A wheat TIP isoform was able to complement a yeast mutant deficient in ammonium transporters,30 suggesting that it is targeted to the yeast plasma membrane. Proteome studies of Arabidopsis plasma membranes12,13 also identified these three proteins. Their identification both in this and the present study provides further support for the view that these proteins are in fact a component of plant plasma membranes, although some contamination of the plasma membranes with tonoplast cannot be ruled out. A TC sequence with 5 predicted TMDs homologous to an ATP/ADP translocator from rice was identified in band 9 and a putative plasma membrane permease was identified in band 6. Five TC sequences corresponding to three classes of oleosins: steroleosin, caleosin, and oleosin (3 isoforms; Table 2) were identified in bands 7, 8, and 9, respectively. Oleosins are small plant proteins characterized by a long hydrophobic core flanked by amphipathic N- and C-terminal domains, which act as emulsifiers for the storage of lipids in seeds.31 The identification of these proteins reflects the specialized role of the aleurone layer, which is an important site for oil storage in cereal seeds and is rich in oil bodies. Oil bodies have been shown to associate closely with plasma membranes during seed desiccation in maize,32 which might explain the presence of oleosins in the aleurone layer plasma membrane fraction. TC sequences corresponding to three isoforms (Table 2) of voltage-dependent anion channels (VDAC) were identified in bands 8 and 9. These proteins form a β-barrel pore in the membrane33 and thus are not predicted to have transmembrane helices by TMPRED (Table 1). These proteins, also known as porins, are present in mitochondrial membranes but have been suggested also to occur in plasma membranes.33 Porins were also identified in Arabidopsis plasma membrane preparations, and this localization was confirmed by transient expression of a GFP fusion in onion cells.12 Barley reticulon was identified in band 9. Reticulon was recently reported to be involved in membrane trafficking between endoplasmic reticulum and Golgi34 but was also identified in Arabidopsis plasma membranes.12 A GTP binding protein β-subunit with no predicted TMDs was also identified in band 9. A similar GTPbinding protein was identified in a plasma membrane preparation from Arabidopsis and was predicted to contain a prenylation site.12 These proteins contain a so-called WD-40 β-propellor motif that is involved in protein-protein interactions. Two previously unknown proteins were identified. A TC sequence encoding a protein predicted to have 4 TMDs that Journal of Proteome Research • Vol. 5, No. 11, 2006 3111
research articles was homologous only to proteins of unknown function was identified in band 4. An NCBI conserved domain (CD) search of the translated TC sequence revealed a domain of unknown function, DUF21, found at the N-terminus of transmembrane proteins between two intracellular domains (CBS domains; pfam00571) with a potential role in dimerization. A TC sequence encoding a protein with 6 predicted TMDs and no homologues of known function was identified in band 9. Two proteins encoded by hypersensitive-reaction-induced genes, HvHIR1 and HvHIR4,35 were identified in band 9 (Tables 1 and 2). Two proteins homologous to these (At1 g69840 and At5 g62740) were found to be enriched in preparations of detergent-resistant plasma membrane lipid rafts from Arabidopsis,36 strongly suggesting that they are indeed plasma membrane associated although they contain no predicted TMDs. These proteins contain a conserved N-terminal prohibitin-like domain and belong to the Proliferation, Ion, and Death (PID) superfamily of plant proteins that are thought to be involved in regulation of ion channels.37
Conclusions A plasma membrane-enriched fraction has been prepared from a challenging plant tissue, the barley seed aleurone layer. This study is, to our knowledge, the first proteome study of seed membranes and the first analysis of intrinsic plasma membrane proteins from an important crop plant. Due to careful analysis, it became apparent that common washing techniques for removal of soluble proteins from membrane fractions were not completely effective for the aleurone layer membranes. An alternative approach based on reversed-phase chromatography on Nucleosil C4 was developed, which resulted in a significant reduction in the amount of soluble contaminants in the membrane protein fraction. Thus, a fraction highly enriched for hydrophobic proteins was obtained allowing a greater proportion of proteins containing several TMDs to be identified in comparison with studies in which standard washing techniques were used. Identifications of several integral membrane proteins were achieved for the first time in barley aleurone layer membranes. The knowledge gained will facilitate future analysis of changes in the aleurone layer membrane proteome during processes related to seed germination. Characterization of membrane proteomes is of great importance for an understanding of cellular functions in all organsims. The method presented here should be evaluated for its potential to enrich for integral membrane proteins. This may be especially relevant for the up and coming “top-down” approaches for membrane proteomics.38
Acknowledgment. We thank Mette. T. Christensen (Technical University of Denmark) for help with aleurone layer preparation and Kate Rafn and Lene Skou (University of Southern Denmark) for help with mass spectrometry. Anja Thoe Fuglsang (Royal Veterinary and Agricultural University, Denmark), Maarten Chrispeels (University of California, San Diego, U.S.A.), Evan Evans (University of Adelaide, Australia), and Jørn Hejgaard (Technical University of Denmark) are thanked for providing antibodies. This work is supported by the Danish Centre for Advanced Food Studies, the Danish Agricultural and Veterinary Research Council (C.F.), and an EMBO short-term fellowship ASTF39-2005 (R.H.). 3112
Journal of Proteome Research • Vol. 5, No. 11, 2006
Hynek et al.
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PR0602850
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