Number Of Transmembrane Domains Research Articles

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

Genome-wide identification and expression analysis of the CLC superfamily genes in tea plants (Camellia sinensis).

The voltage-gated chloride channel (CLC) superfamily is one of the most important anion channels that is widely distributed in bacteria and plants. CLC is involved in transporting various anions such as chloride (Cl-) and fluoride (F-) in and out of cells. Although Camellia sinensis is a hyper-accumulated F plant, there is no studies on the CLC gene superfamily in the tea plant. Here, 8 CLC genes were identified from C. sinensis and they were named CsCLC1-8. The structure of CsCLC genes and the proteins were not conserved; the number of exons varied from 3 to 24, and the number of transmembrane domains contained 2 to 10. Furthermore, phylogenetic analysis revealed that CsCLC4-8 in subclass I contained the typical conserved domains GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), and CsCLC1-3 in subclass II did not contain any of the three conserved residues. We measured the expression levels of CsCLCs in roots, stems and leaves to assess the responses to different concentrations of Cl- and F-. The result indicated that CsCLCs participated in subfunctionalization in response to Cl- and F-, and CsCLC1-3 was more sensitive to F- treatments than CsCLC4-8, CsCLC6 and CsCLC7 may participate in absorption and long-distance transport of Cl-.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

The number of the C-terminal transmembrane domains has the potency to specify subcellular localization of Sec22c

Sec22c has been characterized as an endoplasmic reticulum (ER)-localized transmembrane protein involved in regulation of the vesicle transport between the ER and the Golgi. Sec22c has several isoforms generated by alternative splicing that changes the number of the C-terminal transmembrane domains (TMDs). However, the physiological significance of the splicing remains unknown. Here we show that the splicing isoforms containing four TMDs unexpectedly localized at cis-Golgi, whereas the splicing isoforms containing less than four TMDs localized at the ER. The C-terminal fragment containing the four TMDs was sufficient for the cis-Golgi localization and bound to ADP-ribosylation factor 4 (ARF4). ARF4 knockdown and overexpression of a constitutively active mutant of ARF4 decreased the cis-Golgi localization of the C-terminal fragment and the full-length protein, respectively. These results indicate that the splicing-dependent changes in the number of TMDs allow Sec22c to regulate the subcellular localization in cooperation with ARF4, implying that Sec22c will function at the Golgi as well as the ER.

Molecular characterization of the carbon dioxide receptor in the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae)

The blowfly Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) cannot only act as a mechanical vector of various pathogens, but also infest man and animals causing human health problems and economic losses in the livestock and fish industries. As in other insects, olfaction of this species plays an important role in host location and is presumably mediated via transmembrane receptor signaling pathways. Here, we isolate and characterize CmegGr1 and CmegGr2, two new members of the chemosensory receptor gene family from C. megacephala. The open reading frames of CmegGr1 and CmegGr2 cDNA clones encode 453 and 486 amino acid residues, respectively. These two deduced proteins display high amino acid conservation with previously identified carbon dioxide (CO₂) receptors, such as Drosophila melanogaster Gr21a/Gr63a and Anopheles gambiae s.s. Gr22/Gr24. Further sequence analysis showed that both proteins are consistent with their corresponding orthologs in the membrane topology prediction with some ambiguities in the location of N terminus and the number of transmembrane domains. The transcripts of CmegGr1 and CmegGr2 were detected in the major chemosensory organs including the antennae and proboscises with maxillary palps attached. These results suggest that CmegGr1 and CmegGr2 are likely to be the primary receptors for CO₂ detection in C. megacephala. Knowledge of the molecular identity of the blowfly olfactory CO₂ receptors may aid in the development of novel control strategies designed to take advantage of this unique and critical olfactory pathway.

The path to preservation: Using proteomics to decipher the fate of diatom proteins during microbial degradation

We drew upon recent advances in tandem mass spectrometry‐based proteomic analyses in order to examine the proteins that remain after a diatom bloom enters the stationary phase, precipitates out of the photic zone, and is subjected to microbial degradation over a 23‐d period within a controlled laboratory environment. Proteins were identified from tandem mass spectra searched against three different protein databases in order to track proteins from Thalassiosira pseudonana and any potential bacterial contributions. A rapid loss of diatom protein was observed over the incubation period; 75% of the proteins initially identified were not detected after 72 h of exposure to a microbial population. By the 23rd day, peptides identified with high confidence correlated with only four T. pseudonana proteins. Five factors may have influenced the preservation of diatom proteins: (1) protection within organelles or structures with multiple membranes, (2) the relative cellular abundance in the photosynthetic apparatus, (3) the number of transmembrane domains in the protein sequence, (4) the presence of glycan modification motifs, and (5) the capability of proteins or peptides to aggregate into supramolecules.

Identification and characterization of a novel bacterial ATP-sensitive K+ channel

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K(+) (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K(+) selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K(+) channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir's compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K(+) current with characteristics similar to those of the ATP-sensitive K(+) (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.

Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents.

Somatostatin and cortistatin exert multiple biological actions through five receptors (sst1-5); however, not all their effects can be explained by activation of sst1-5. Indeed, we recently identified novel truncated but functional human sst5-variants, present in normal and tumoral tissues. In this study, we identified and characterized three novel truncated sst5 variants in mice and one in rats displaying different numbers of transmembrane-domains [TMD; sst5TMD4, sst5TMD2, sst5TMD1 (mouse-variants) and sst5TMD1 (rat-variant)]. These sst5 variants: (1) are functional to mediate ligand-selective-induced variations in [Ca(2+)]i and cAMP despite being truncated; (2) display preferential intracellular distribution; (3) mostly share full-length sst5 tissue distribution, but exhibit unique differences; (4) are differentially regulated by changes in hormonal/metabolic environment in a tissue- (e.g., central vs. systemic) and ligand-dependent manner. Altogether, our results demonstrate the existence of new truncated sst5-variants with unique ligand-selective signaling properties, which could contribute to further understanding the complex, distinct pathophysiological roles of somatostatin and cortistatin.

Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155

BackgroundThe usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis.ResultsA proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. smegmatis. An enzymatic cell surface shaving method was used to determine the surface-exposed proteins. As a result, a total of 390 cell wall proteins and 63 surface-exposed proteins were identified. Further analysis of the 390 cell wall proteins provided the theoretical molecular mass and pI distributions and determined that 26 proteins are shared with the surface-exposed proteome. Detailed information about functional classification, signal peptides and number of transmembrane domains are given next to discussing the identified transcriptional regulators, transport proteins and the proteins involved in lipid metabolism and cell division.ConclusionIn short, a comprehensive profile of the M. smegmatis cell wall subproteome is reported. The current research may help the identification of some valuable vaccine and drug target candidates and provide foundation for the future design of preventive, diagnostic, and therapeutic strategies against mycobacterial diseases.

NTPDase family in zebrafish: Nucleotide hydrolysis, molecular identification and gene expression profiles in brain, liver and heart

The nucleoside triphosphate diphosphohydrolase (NTPDase) family cleaves tri- and diphosphonucleosides to monophosphonucleosides and is responsible for terminating purinergic transmission. Since the NTPDase family in zebrafish is poorly understood, here we evaluated the nucleotide hydrolysis in three tissues of adult zebrafish (brain, liver, and heart), confirmed the presence of distinct NTPDase members by a phylogenetic analysis and verified their relative gene expression profiles in the respective tissues. A different profile of ATP and ADP hydrolysis in the brain, liver, and heart as a function of time and protein concentration was observed. Sodium azide (20mM), ARL 67156 (300 microM) and Suramin (300 microM) differently altered the nucleotide hydrolysis in zebrafish tissues, suggesting the contribution of distinct NTPDase activities. Homology-based searches identified the presence of NTPDase1-6 and NTPDase8 orthologs and the phylogeny also grouped three NTPDase2 and two NTPDase5 paralogs. The deduced amino acid sequences share the apyrase conserved regions, conserved cysteine residues, putative N-glycosylation, phosphorylation, N-acetylation sites, and different numbers of transmembrane domains. RT-PCR experiments revealed the existence of a distinct relative entpd1-6 and entpd8 expression profile in brain, liver, and heart. Taken together, these results indicate that several NTPDase members might contribute to a tight regulation of nucleotide hydrolysis in zebrafish tissues.

Identification of a Disulfide Bridge Essential for Transport Function of the Human Proton-coupled Amino Acid Transporter hPAT1

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG(11)-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.

Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of approximately 140-kDa was observed, which probably corresponds to a monomeric SecYEG-substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.

Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.

MLC1: A Novel Protein in Distal Astroglial Processes

Megaloencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease in children caused by mutations in the MLC1 gene. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation. MLC1 encodes a novel protein, MLC1, which is mainly expressed in the brain and leukocytes. The function is unknown, although a transport function has been suggested. In this article, we provide experimental data addressing the membrane topology and cellular localization of MLC1. We show that MLC1 contains an even number of transmembrane domains, supporting the possible transport function of MLC1. We demonstrate that MLC1 is specifically expressed in distal astroglial processes in perivascular, subependymal, and subpial regions. This localization suggests a role for MLC1 in a transport process across the blood-brain and brain-cerebrospinal fluid barriers. Astrocyte functions have long been debated. It is becoming increasingly clear that these cells are of fundamental importance in maintaining the structural and functional integrity of neural tissue. Elucidation of the function of MLC1 will contribute to a better understanding of not only the pathophysiology of the disease, but also the role of astrocytes in normal neural tissue.

Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains.

The solute carrier gene family SLC26 consists of tissue-specific anion exchanger genes, three of them associated with distinct human recessive disorders. By a genome-driven approach, several new SLC26 family members have been identified, including a kidney- and pancreas-specific gene, SLC26A6. We report the functional characterization of SLC26A6 and two new alternatively spliced variants, named SLC26A6c and SLC26A6d. Immunofluorescence studies on transiently transfected cells indicated membrane localization and indicated that both NH(2)- and COOH-terminal tails of the SLC26A6 variants are located intracellularly, suggesting a topology with an even number of transmembrane domains. Functional expression of the three proteins in Xenopus oocytes demonstrated Cl(-) and SO(4)(2-) transport activity. In addition, the transport of SO(4)(2-) and Cl(-) was inhibited by DIDS and HCO(3)(-). We demonstrated also that the COOH terminus of SLC26A6 binds to the first and second PDZ domains of the Na(+)/H(+) exchanger (NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor (NHERF) proteins in vitro. Truncation of the last three amino acids (TRL) of SLC26A6 abrogated the interaction but did not affect transport function. These results demonstrate that SLC26A6 and its two splice variants can function as anion transporters linked to PDZ-interaction pathways. Our results support the general concept of microdomain organization for ion transport and suggest a mechanism for cystic fibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulation in pancreatic duct cells.

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

C-terminal topology of gastric H+,K(+)-ATPase.

An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K(+)-ATPase. The antibody bound to a 95 kDa band of H+,K(+)-ATPase that was solubilized in SDS, but not to that of Na+,K(+)-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K(+)-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K(+)-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K(+)-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K(+)-ATPase alpha-subunit has an even number of transmembrane domains.

Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases.

Complementary DNAs for two isoforms of the plasma membrane Ca2+-ATPase from rat brain have been isolated. The cDNAs were identified using an oligonucleotide probe derived from a conserved amino acid sequence of the ATP binding site of the aspartylphosphate family of transport ATPases. The complete nucleotide sequences have been determined, and the primary structures for both isoforms have been deduced. The Mr of isoform 1, which has 1,176 amino acids, is 129,500 and that of isoform 2, which has 1,198 amino acids, is 132,605. A region of isoform 1 exhibits perfect amino acid identity with a fragment consisting of 17 amino acids from the phosphorylation domain of the human erythrocyte calmodulin-sensitive Ca2+-ATPase (James, P., Zvaritch, E.I., Shakhparonov, M.I., Penniston, J.T., and Carafoli, E. (1987) Biochem. Biophys. Res. Commun. 149, 7-12). A comparison of transport ATPases from diverse species has allowed the identification of a sequence that may form part of a second ATP binding site. Amino acid similarity and hydropathy profile comparisons suggest that the transmembrane organization of the plasma membrane Ca2+-ATPase is the same as that of the Na+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase; the data indicate that each of these enzymes has an even number of transmembrane domains and that their C termini are located on the cytoplasmic side of the membrane. An arginine-rich sequence that is highly characteristic of the "A" domain of a calmodulin binding site is located near the C termini of both isoforms. This putative A domain is identical in isoforms 1 and 2 but their "B" domains, and the remaining C-terminal sequences, are very different. However, 3'-untranslated sequences of the isoform 1 transcript have the potential to encode a calmodulin binding B domain and a C terminus that is very similar to that of isoform 2. This suggests the possibility that additional diversity may occur via alternative processing of the primary transcript.