In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.     

The human platelet membrane proteome reveals several new potential membrane proteins

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.     

Identification of a residue in helix 2 of rice plasma membrane intrinsic proteins that influences water permeability

Molecular selection, ion exclusion, and water permeation are well known regulatory mechanisms in aquaporin. Water permeability was found to be diverse in different subgroups of plasma membrane intrinsic proteins (PIPs), even though the residues surrounding the water holes remained the same across the subgroups. Upon homology modeling and structural comparison, a conserved Ala/Ile(Val) residue difference was identified in helix 2 that affected the conformation of the NPA region and consequently influenced the water permeability. The residue difference was found to be conservative within the two subgroups of PIPs in rice as well as in other plants. Functional tests further confirmed the prediction via site-directed mutagenesis where replacement of Ala(103) or Ala(102) in respective OsPIP1;1 or OsPIP1;3 with Val yielded 7.0- and 2.2-fold increases in water transportation, and substitution of Ile(98) or Val(95) in respective OsPIP2;3 or OsPIP2;7 with Ala resulted in 73 or 52% reduction of water transportation. Based on structural analyses and molecular dynamics simulations, we proposed that the difference in water permeability was attributed to the orientation variations of helix 2 that modified water-water and water-protein interactions.     

Plasma membrane proteome in Arabidopsis and rice

Plant cells contain many membrane systems that are specially adapted to perform particular functions. In plant cells, the processing of signals that are involved in responses to biotic and abiotic stressors occurs in the plasma membrane. Therefore, characterization of the plasma membrane proteome can provide new insights into the functions of various plant membrane systems. Plant plasma membrane proteomics can also provide valuable information for plant-specific biological investigations. Despite recent advances in preparative and analytical techniques for plant plasma membrane proteins, the characterization of these proteins, particularly the hydrophobic ones, remains challenging. In this review, plant plasma membrane proteomics data compiled from the literature on Arabidopsis thaliana are presented. Initial attempts to determine the physiological significance of some proteins identified from plasma membrane proteomics in rice and other plants are also described from the results of our research.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Crystallization of intrinsic membrane proteins

After many years of discouraging failures, it is now possible to crystallize the intrinsic membrane proteins and to obtain structural information from diffraction studies on the crystals. The strategy for the crystallization consists of depletion of boundary phospholipids from the protein and complex formation with specific ligands.     

Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis

Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.     

Simplified enrichment of plasma membrane proteins for proteomic analyses in Arabidopsis thaliana

The plasma membrane (PM) serves as the point of contact between cells and the outside environment. As such, changes in the PM proteome are an important component of understanding cellular responses to a diverse array of stimuli. However, intricate sample handling to enrich PM proteomes by traditional methods is both technically challenging and time consuming. Here, we describe a simplified method for decreasing the representation of other membrane-containing organelles such as the endoplasmic reticulum, plastids and mitochondria from crude microsomal membrane isolations. The decrease in other organellar proteomes results in an increase in both the total number of PM proteins and the number of spectra identified from these proteins representing the PM proteome. Therefore, this strategy represents a simple and rapid method for enriching PM proteins from Arabidopsis cell cultures for proteomic analyses.     

Use of a proteome strategy for tagging proteins present at the plasma membrane

A plasma membrane (PM) fraction was purified from Arabidopsis thaliana using a standard procedure and analyzed by two-dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty-two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM-specific and PM-enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM-specific spots correspond to proteins with unknown function. Among the later, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of Arabidopsis genome sequencing, for genome-wide exploration of plant function.     

Estimation of membrane proteins in the human proteome

Genomics and proteomics have added valuable information to our knowledgebase of the human biological system including the discovery of therapeutic targets and disease biomarkers. However, molecular profiling studies commonly result in the identification of novel proteins of unknown localization. A class of proteins of special interest is membrane proteins, in particular plasma membrane proteins. Despite their biological and medical significance, the 3-dimensional structures of less than 1% of plasma membrane proteins have been determined. In order to aid in identification of membrane proteins, a number of computational methods have been developed. These tools operate by predicting the presence of transmembrane segments. Here, we utilized five topology prediction methods (TMHMM, SOSUI, waveTM, HMMTOP, and TopPred II) in order to estimate the ratio of integral membrane proteins in the human proteome. These methods employ different algorithms and include a newly-developed method (waveTM) that has yet to be tested on a large proteome database. Since these tools are prone for error mainly as a result of falsely predicting signal peptides as transmembrane segments, we have utilized an additional method, SignalP. Based on our analyses, the ratio of human proteins with transmembrane segments is estimated to fall between 15% and 39% with a consensus of 13%. Agreement among the programs is reduced further when both a positive identification of a membrane protein and the number of transmembrane segments per protein are considered. Such a broad range of prediction depends on the selectivity of the individual method in predicting integral membrane proteins. These methods can play a critical role in determining protein structure and, hence, identifying suitable drug targets in humans.     

植物质膜水通道蛋白转运及逆境胁迫响应的分子调控机制

质膜水通道蛋白即质膜内在蛋白(plasma membrane intrinsic proteins, PIPs),属于通道蛋白,定位在质膜上,是植物体内水分子、CO₂及其他一些小分子溶质跨细胞膜运输的通道。PIPs对运输基质具有高度选择性,在维持植物细胞的水分平衡过程中发挥重要作用。PIPs的表达、活性与定位不但受转录水平和翻译后水平的调控,而且受外界环境影响。研究表明在非生物胁迫下,PIPs表达模式和定位会发生改变。本文重点阐述了PIPs转运的分子机制、转录水平及翻译后水平的调控机制以及PIPs对非生物胁迫的响应机制,分析了目前关于PIPs的研究动态和值得探究的研究方向,以期帮助相关领域的科研人员对PIPs的研究进展有更深入地了解。     

Identification of GTP-binding proteins in the plasma membrane of higher plants

Antisera raised against a highly conserved amino acid sequence (G alpha-common peptide) of animal Gs alpha, Gi alpha, Go alpha and Gt alpha recognize, in plasma membranes of several higher plants, sets of proteins of Mr = 37 and 31 kDa (Vicia faba), 36 and 31 kDa (Arabidopsis thaliana) and 38 and 34 kDa (Commelina communis). The A. thaliana proteins were solubilized and partially purified. They bind [35S]GTP gamma S with high affinity (apparent Kd approximately 10 nM) and, with lower affinity, GTP but not the other nucleotides tested (ATP, CTP, ITP, UTP).     

The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.     

Identification of ubiquitinated proteins in Arabidopsis

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.     

Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.     

Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.     

Identification of calcium-binding proteins associated with the lymphocyte plasma membrane

The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr which are solubilised by Ca2+-chelators. As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr "calelectrin" of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.     

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.     

Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana

MOTIVATION: The completion of the Arabidopsis genome offers the first opportunity to analyze all of the membrane protein sequences of a plant. The majority of integral membrane proteins including transporters, channels, and pumps contain hydrophobic alpha-helices and can be selected based on TransMembrane Spanning (TMS) domain prediction. By clustering the predicted membrane proteins based on sequence, it is possible to sort the membrane proteins into families of known function, based on experimental evidence or homology, or unknown function. This provides a way to identify target sequences for future functional analysis. RESULTS: An automated approach was used to select potential membrane protein sequences from the set of all predicted proteins and cluster the sequences into related families. The recently completed sequence of Arabidopsis thaliana, a model plant, was analyzed. Of the 25,470 predicted protein sequences 4589 (18%) were identified as containing two or more membrane spanning domains. The membrane protein sequences clustered into 628 distinct families containing 3208 sequences. Of these, 211 families (1764 sequences) either contained proteins of known function or showed homology to proteins of known function in other species. However, 417 families (1444 sequences) contained only sequences with no known function and no homology to proteins of known function. In addition, 1381 sequences did not cluster with any family and no function could be assigned to 1337 of these.