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Peripheral Membrane Protein (peripheral + membrane_protein)
Selected AbstractsNovel interactors and a role for supervillin in early cytokinesis,CYTOSKELETON, Issue 6 2010Tara C. Smith Abstract Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle. © 2010 Wiley-Liss, Inc. [source] Characterization of CetA and CetB, a bipartite energy taxis system in Campylobacter jejuniMOLECULAR MICROBIOLOGY, Issue 5 2008Kathryn T. Elliott Summary The energy taxis receptor Aer, in Escherichia coli, senses changes in the redox state of the electron transport system via an flavin adenine dinucleotide cofactor bound to a PAS domain. The PAS domain (a sensory domain named after three proteins Per, ARNT and Sim, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors. An apparent energy taxis system in Campylobacter jejuni is composed of two proteins, CetA and CetB, that have the domains of Aer divided between them. CetB has a PAS domain, while CetA has a predicted transmembrane region, HAMP domain and the HCD. In this study, we examined the expression of cetA and cetB and the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane. CetB levels are CetA dependent. Additionally, we demonstrated that both CetA and CetB participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB. [source] Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteinsPLANT BIOLOGY, Issue 5 2010A. Campos Abstract Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo-adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C-terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co-localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light-induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two-dimensional gel electrophoresis discriminated, for the first time, the 58-kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC-MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection. [source] Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extractsCYTOSKELETON, Issue 10 2007Ana S. DePina Abstract Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans -Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of ,0.30 ,m/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 ,m/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Defining the membrane proteome of NK cellsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2010Dhimankrishna Ghosh Abstract The present study was initiated to define the composition of the membrane proteome of the Natural Killer (NK) like cell line YTS. Isolated membranes were treated with reagents that have been reported to remove peripheral membrane proteins. Additional steps involving trifluoroethanol (TFE) were introduced in an effort to remove remaining nonintegral membrane proteins. This treatment resulted in the release of a subset of proteins without any apparent disruption of membrane integrity. The membranes were solubilized and digested with trypsin in 25% TFE. The resulting peptides were separated using an off-line two-dimensional reversed phase LC technique at alkaline and acidic pHs. Mass spectrometric analysis identified 1843 proteins with high confidence scores. On the basis of the presence of transmembrane regions or evidence of posttranslational modifications and prediction algorithms, approximately 40% of the identified proteins were predicted as plausible membrane proteins. The remaining species were largely involved in cellular processes and molecular functions that could be predicted to be transiently associated with membranes. The analytical approaches presented in this study offer robust generic methods for the identification and characterization of membrane proteins. These observations highlight the fact that the membrane is a dynamic entity that is composed of integral and stably associated proteins. Copyright © 2009 John Wiley & Sons, Ltd. [source] Advances in membrane receptor screening and analysisJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2004Matthew A. Cooper Abstract During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand,membrane,receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included. Copyright © 2004 John Wiley & Sons, Ltd [source] | |||||||||||||