Synthesis and assembly of the membrane proteins in E. coli.

Kinetics of integration of membrane proteins were studied in E. coli to discover how membrane proteins find their final location in the functional membrane. The experiments make use of a simple and convenient method developed for isolating inner and outer membranes from a number of small-scale cultures with high recovery. Among the proteins that constitute the cell surface structures, inner membrane proteins are integrated most rapidly after synthesis, whereas outer membrane proteins delay somewhat, and periplasmic proteins delay further in reaching their destinations. Protein I, a major outer membrane protein with molecular weight of about 37,000 daltons, exhibits significantly slower rates of integration than other outer membrane proteins. The decreased fluidity of membrane lipids by temperature shiftdown of an unsaturated fatty acid auxotroph grown on elaidate results in abnormally slow assembly of the outer membrane proteins and also in an anomalous assembly of the inner membrane proteins, suggesting that the fluid state of the lipids is required for normal operation of these processes. The possible relevance of these findings to the mechanism of membrane formation is discussed.     

A simple statistical method for discriminating outer membrane proteins with better accuracy

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have systematically analyzed the amino acid composition of globular proteins from different structural classes and outer membrane proteins. We found that the residues, Glu, His, Ile, Cys, Gln, Asn and Ser, show a significant difference between globular and outer membrane proteins. Based on this information, we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 89% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 80%. These accuracy levels are comparable to other methods in the literature, and this is a simple method, which could be used for dissecting outer membrane proteins from genomic sequences. The influence of protein size, structural class and specific residues for discrimination is discussed.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis.

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007--4021) has been modified for the separation of Acholeplasma laidlawii proteins. Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein. A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100. Exterior-facing membrane proteins were distinguished from the interior-facing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinating intact cells. A. laidlawii was also grown in the presence of NaH232PO4 and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.     

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Rampant Exchange of the Structure and Function of Extramembrane Domains between Membrane and Water Soluble Proteins

Of the membrane proteins of known structure, we found that a remarkable 67% of the water soluble domains are structurally similar to water soluble proteins of known structure. Moreover, 41% of known water soluble protein structures share a domain with an already known membrane protein structure. We also found that functional residues are frequently conserved between extramembrane domains of membrane and soluble proteins that share structural similarity. These results suggest membrane and soluble proteins readily exchange domains and their attendant functionalities. The exchanges between membrane and soluble proteins are particularly frequent in eukaryotes, indicating that this is an important mechanism for increasing functional complexity. The high level of structural overlap between the two classes of proteins provides an opportunity to employ the extensive information on soluble proteins to illuminate membrane protein structure and function, for which much less is known. To this end, we employed structure guided sequence alignment to elucidate the functions of membrane proteins in the human genome. Our results bridge the gap of fold space between membrane and water soluble proteins and provide a resource for the prediction of membrane protein function. A database of predicted structural and functional relationships for proteins in the human genome is provided at sbi.postech.ac.kr/emdmp.     

Organization of lymphocyte plasma membrane. Surface protein-membrane matrix interactions in B-cell lines of different stages of differentiation

Composition of surface proteins and their interactions with cytoskeleton or membrane matrix were compared in tumor B-cell lines of different stages of B-lymphocyte maturation. All studied B-cell lines were found to share a similar set of cell surface proteins, which are tightly associated with the cytoskeleton. The increase in amount of detergent-unextractable cell surface proteins with B-cell maturation suggested that differentiation of B lymphocytes was accompanied by development of specific interactions between surface proteins and elements of the cytoskeleton or membrane matrix. Using a recently developed procedure for lymphocyte plasma membrane fractionation we demonstrate changes in distribution of cell surface proteins in membrane matrix-rich and membrane matrix-poor plasma membrane fractions during B-lymphocyte maturation. Thus, cell surface proteins of the mature B-cell line MOPC-315 were predominantly found in the plasma membrane vesicles of a high buoyant density. These vesicles mostly contained plasma membrane proteins tightly associated with elements of the membrane matrix. In immature B cells (line 70Z3) virtually all surface proteins were detected in both low and high buoyant density membrane vesicles. The tendency to increased associations between surface proteins and cytoskeleton/membrane matrix with maturation of B cells could not be explained by increased amounts of filamentous actin, since no correlation was found between the amount of globular or filamentous actin and the degree of surface protein-cytoskeleton (membrane matrix) interactions.     

鼻咽癌5-8F细胞膜蛋白质组初步分析

细胞膜蛋白是细胞的重要组成部分,作为细胞的"门铃"与"门户",参与细胞内外物质交换、信息转换、细胞生长发育、细胞迁移以及免疫应答等重要生理活动.为鉴定鼻咽癌转移相关膜蛋白,运用差速离心联合双水相方法分离纯化鼻咽癌高转移细胞5-8F的细胞膜,SDS-PAGE分离膜蛋白,液相色谱/电喷雾串联质谱分析(LC-MS/MS)结合生物信息学分析鉴定出316种非冗余蛋白质,其中152种(48.5%)被注释为膜蛋白或膜相关蛋白.通过肿瘤差异蛋白质组数据库(dbDEPD)搜索,发现在114个膜蛋白中有49种膜蛋白与其它肿瘤的发生发展密切相关,其中21个膜蛋白与肿瘤转移相关.进一步分析发现膜蛋白CD104、VDAC2、CD298和SLC25A3与同属头颈部肿瘤的口腔癌转移相关,提示这4个膜蛋白也可能是鼻咽癌潜在的转移相关蛋白.研究结果提供了一个鼻咽癌细胞5-8F包含中高丰度膜蛋白的数据库,为进一步研究头颈部肿瘤鼻咽癌癌变分子机理积累了有价值的资料.     

LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.     

Membrane protein solubilization: recent advances and challenges in solubilization of serotonin1A receptors

Solubilization of integral membrane proteins is a process in which the proteins and lipids that are held together in native membranes are suitably dissociated in a buffered detergent solution. The controlled dissociation of the membrane results in formation of small protein and lipid clusters that remain dissolved in the aqueous solution. Effective solubilization and purification of membrane proteins, especially heterologously-expressed proteins in mammalian cells in culture, in functionally active forms represent important steps in understanding structure-function relationship of membrane proteins. In this review, critical factors determining functional solubilization of membrane proteins are highlighted with the solubilization of the serotonin 1A receptor taken as a specific example.     

The role of the transmembrane domain in determining the targeting of membrane proteins to either the inner envelope or thylakoid membrane

Chloroplastic membrane proteins can be targeted to any of three distinct membrane systems, i.e., the outer envelope membrane (OEM), inner envelope membrane (IEM), and thylakoid membrane. This complex structure of chloroplasts adds significantly to the challenge of studying protein targeting to various membrane sub-compartments within a chloroplast. In this investigation, we examined the role played by the transmembrane domain (TMD) in directing membrane proteins to either the IEM or thylakoid membrane. Using the IEM protein, Arc6 (Accumulation and Replication of Chloroplasts 6), we exchanged the stop-transfer TMD of Arc6 with various TMDs derived from different IEM and thylakoid membrane proteins and monitored the subcellular localization of these Arc6-hybrid proteins. We showed that when the Arc6 TMD was replaced with a TMD derived from various thylakoid membrane proteins, these Arc6(thylTMD) hybrid proteins could be directed to the thylakoid membrane rather than to the IEM. Conversely, when the TMD of the thylakoid membrane proteins, STN8 (State Transition protein kinase 8) or Plsp1 (Plastidic type I signal peptidase 1), was replaced with the stop-transfer TMD of Arc6, STN8 and Plsp1 were halted at the IEM. From our investigation, we conclude that the TMD plays a critical role in targeting integral membrane proteins to either the IEM or thylakoid membrane.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Forces and factors that contribute to the structural stability of membrane proteins

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.     

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

A novel method for isolation of membrane proteins: a baculovirus surface display system

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins. In the first method, we isolated proteins directly from the envelope of budding BmNPV virions. In the second method, we isolated proteins from cellular membranes that had disintegrated due to viral egress. We isolated 6756 proteins. Of these, 1883 have sequence similarities to membrane proteins and 1550 proteins are homologous to known membrane proteins. This study indicates that membrane proteins can be effectively isolated using our BVSD system. Using an analogous method, membrane proteins can be isolated from other eukaryotic organisms, including human beings, by employing a host cell-specific budding virus.     

Interplay between the electrostatic membrane potential and conformational changes in membrane proteins

Transmembrane electrostatic membrane potential is a major energy source of the cell. Importantly, it determines the structure as well as function of charge‐carrying membrane proteins. Here, we discuss the relationship between membrane potential and membrane proteins, in particular whether the conformation of these proteins is integrally connected to the membrane potential. Together, these concepts provide a framework for rationalizing the types of conformational changes that have been observed in membrane proteins and for better understanding the electrostatic effects of the membrane potential on both reversible as well as unidirectional dynamic processes of membrane proteins.     

Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE, and LC-MS/MS

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.