DOCSLIB.ORG

Types of Membrane Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Types of Membrane Proteins All cells have membranes eukaryotic cells have intracellular membranes • Organelles with 2 • Organelles with a Molecular Membranes or more single membrane Biology membranes • endoplasmic • nucleus (2) reticulum • mitochondria (2) • golgi complex • chloroplasts (3) • endosomes • peroxisomes • lysosomes Types of Membrane Proteins • Integral membrane proteins • Lipid-linked proteins • Peripheral membrane proteins Integral Membrane Proteins • 20 - 30% of total proteins • 60% of drug targets • Essential functions – Transfer information – Transport material – Energy conversion • Few Structures available (~100 helical membrane proteins) Alberts, Molecular Biology of the Cell (1994) p.486. Integral Membrane Protein difficulties: Membrane protein topology • Low abundance • Number of transmembrane segments • Instability outside their native membranes • Their orientation relative to the membrane • Inefficient refolding • Ability to grow ordered 2- or 3-D crystals Advances have largely been made with bacterial proteins Hydrophobicity Scale Membrane protein Explorer ‘Mpex’ http://blanco.biomol.uci.edu/mpex/ Branden & Tooze, p. 245. MacKinnon (2005) Science, pp. 1425 - 1426 Hydrophobicity plots for chains L & M of Reaction center Rhodocater sphaeroides 19 aa window Kyte & Doolittle Hydrophobicity scale Branden & Tooze p.246 Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Fig. 1. Topology mapping using the HA/Suc2/His4C reporter Kim, Hyun et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11142-11147 TIBS (2000) 25:433 Copyright ©2006 by the National Academy of Sciences Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Test of Membrane topology Bacteriorhodopsin Bovine Ca2+-ATPase • Antibody Epitopes • Proteolysis • Modification – Chemical E. coli ClC Cl-/H+ antiporter6 – Glycosylation Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Hessa, T. Nature 2005 Jan 27;433(7024):377-81. TYPE A SURFACTANTS Lodish, Molecular Cell Biology (2000) p.84 Helenius & Simons (1975) BBA 415:29-79 Type A surfactants (continued) Helenius & Simons (1975) BBA 415:29-79 Helenius & Simons (1975) BBA 415:29-79 GEL FILTRATION Lipid composition of membrane preps (%) -1 Rs = ao + bo (erf )(1-Kd) Rs = Stokes’ radius = constants of gel matrix used ao, bo erf-1 = inverse error function = partial coefficient= (V -V )/V -V ) Kd e 0 t 0 where Ve, V0 and Vt are elution times protein, void and total volume of column, respectively. SUCROSE GRADIENT CENTRIFUGATION AND MOLECULAR WEIGHT . M* = s20,w RT/D20,w (1-v* ps) = sedimentation coefficient, normalized to 20˚C and water Rate-zonal s20,w Centrifugation D = diffusion constant of complex, normalized to 20˚C and 20,w water, can be measured in an analytical ultracentrifuge or it may be replaced by the following expression with Stokes’ radius Rs as a parameter: D= kT/6!nRs where n viscocity of the solvent v* = partial specific volume of complex ps = density of the solvent = gas constant, absolute temperature Lodish, Molecular Cell Biology (2000) p.86 R,T Detergent solubilization of integral membrane proteins Photosynthesis rxn center Purple bacterium Caffrey (2000) Cur. Op. Struct. Biol. 10:486 Branden & Tooze p.237 MacKinnon (2003) Febs Letters KcsA potassium channel from the bacterium Streptomyces lividans 555: 62-65 MacKinnon (2003) Febs Letters 555: 62-65 Integral Membrane Protein Folding Bacteriorhodopsin 1st membrane protein to be completely denatured and refolded in vitro (London & Khorana JBC (1982) 257:7003). Synthetic peptides 3 21-residue peptides that form transmembrane alpha-helices can aggregate/assemble and conduct ions (Lear, Wasserman & DeGrado Science (1989) 240:1437 KcsA MthK (Ca++) MacKinnon (2003) Febs Letters 555: 62-65 Bacteriorhodopsin Folding Models Engelman et al., (2003) FEBS letters 555:122-125 Curran 1999 Transmembrane Regions Integral membrane proteins "-Helices • Type I Single transmembrane span, N- terminus in the ectodomain, C- terminus in the cytosol • Type II Single span, C-terminus in the ectodomain, N-terminus in the cytosol #-barrels • Type III Multiple spans porin from the outer membrane of Rhodobacter capsulatus.
Load more

Recommended publications
  • Membrane Protein Stabilization Strategies for Structural and Functional Studies
    membranes Review Membrane Protein Stabilization Strategies for Structural and Functional Studies Ekaitz Errasti-Murugarren 1,2,*, Paola Bartoccioni 1,2 and Manuel Palacín 1,2,3,* 1 Laboratory of Amino acid Transporters and Disease, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028 Barcelona, Spain; [email protected] 2 CIBERER (Centro Español en Red de Biomedicina de Enfermedades Raras), 28029 Barcelona, Spain 3 Department of Biochemistry and Molecular Biomedicine, Universitat de Barcelona, 08028 Barcelona, Spain * Correspondence: [email protected] (E.E.-M.); [email protected] (M.P.) Abstract: Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies. Keywords: membrane proteins; stability; mutagenesis; detergent; lipid; antibody; nanobody; ligand Citation: Errasti-Murugarren, E.; Bartoccioni, P.; Palacín, M. Membrane Protein Stabilization Strategies for Structural and Functional Studies.
    [Show full text]
  • Snapshot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W
    SnapShot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W. Ng Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604 1230 Cell 129, June 15, 2007 ©2007 Elsevier Inc. DOI 10.1016/j.cell.2007.06.005 See online version for legend and references. SnapShot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W. Ng Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604 Arrows specify the routes of individual pathways. All pathways culminate in substrate degradation by the 26S proteasome. (Left) ERAD Pathways in Budding Yeast (A) Newly synthesized secretory and membrane proteins enter the ER through the Sec61 protein-conducting channel complex unfolded. Hsp70-related molecular chaperones (Kar2p) bind to nascent polypeptides in the ER lumen and to the cytosolic domains of membrane proteins (Hsp70, B). These factors assist in substrate folding and also assist in their disposal if they fail to fold. Mannose residues on misfolded glycoproteins are trimmed by the ER mannosidase Mns1p (E). Mannose trimming facilitates the recognition of misfolded glycoproteins by luminally oriented lectin factors Htm1p and Yos9p. (C, D, and F) At least two ER membrane-localized E3 ubiquitin ligases organize protein complexes that receive and process misfolded proteins. These complexes define three pathways that recognize lesions in the cytosolic (ERAD-C), transmembrane (ERAD-M), and luminal (ERAD-L) domains of substrates. Both ERAD-M and ERAD-L use the Hrd1 ubiquitin ligase but the luminal factor Yos9p is dispensable for ERAD-M. A Hrd1 complex lacking Yos9p has been observed suggesting dedicated complexes for all three pathways.
    [Show full text]
  • Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance
    biomolecules Review Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance Dina Frani´c †, Klara Zubˇci´c † and Mirta Boban * Croatian Institute for Brain Research, School of Medicine, University of Zagreb, 10000 Zagreb, Croatia; [email protected] (D.F.); [email protected] (K.Z.) * Correspondence: [email protected] † Equal contribution. Abstract: Protein homeostasis, or proteostasis, is crucial for the functioning of a cell, as proteins that are mislocalized, present in excessive amounts, or aberrant due to misfolding or other type of damage can be harmful. Proteostasis includes attaining the correct protein structure, localization, and the for- mation of higher order complexes, and well as the appropriate protein concentrations. Consequences of proteostasis imbalance are evident in a range of neurodegenerative diseases characterized by protein misfolding and aggregation, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. To protect the cell from the accumulation of aberrant proteins, a network of protein quality control (PQC) pathways identifies the substrates and direct them towards refolding or elimination via regulated protein degradation. The main pathway for degradation of misfolded proteins is the ubiquitin-proteasome system. PQC pathways have been first described in the cytoplasm and the endoplasmic reticulum, however, accumulating evidence indicates that the nucleus is an important PQC compartment for ubiquitination and proteasomal degradation of not only nuclear, but also cyto- plasmic proteins. In this review, we summarize the nuclear ubiquitin-proteasome pathways involved in proteostasis maintenance in yeast, focusing on inner nuclear membrane-associated degradation (INMAD) and San1-mediated protein quality control. Keywords: proteasome; ubiquitin; nucleus; inner nuclear membrane; yeast; proteostasis; protein quality control; protein misfolding Citation: Frani´c,D.; Zubˇci´c,K.; Boban, M.
    [Show full text]
  • How Does Protein Zero Assemble Compact Myelin?
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 May 2020 doi:10.20944/preprints202005.0222.v1 Peer-reviewed version available at Cells 2020, 9, 1832; doi:10.3390/cells9081832 Perspective How Does Protein Zero Assemble Compact Myelin? Arne Raasakka 1,* and Petri Kursula 1,2 1 Department of Biomedicine, University of Bergen, Jonas Lies vei 91, NO-5009 Bergen, Norway 2 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Aapistie 7A, FI-90220 Oulu, Finland; [email protected] * Correspondence: [email protected] Abstract: Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin – the lipid-rich, periodic structure that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes together via homophilic adhesion, forming a dense, macroscopic ultrastructure known as the intraperiod line. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs the formation of myelin. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when is P0 trafficked and modified to enable myelin compaction, and how disease mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.
    [Show full text]
  • Membrane Proteins Are Associated with the Membrane of a Cell Or Particular Organelle and Are Generally More Problematic to Purify Than Water-Soluble Proteins
    Strategies for the Purification of Membrane Proteins Sinéad Marian Smith Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Ireland. Email: [email protected] Abstract Although membrane proteins account for approximately 30 % of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilization and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non- covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction require phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilization, where problems are most frequently are encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of downstream purification techniques. Key Words: peripheral membrane protein, integral membrane protein, detergent, protein purification, protein solubilization. 1. Introduction Membrane proteins are associated with the membrane of a cell or particular organelle and are generally more problematic to purify than water-soluble proteins. Membrane proteins represent approximately 30 % of the open-reading frames of an organism’s genome (1-4), and play crucial roles in basic cell functions including signal transduction, energy production, nutrient uptake and cell-cell communication.
    [Show full text]
  • Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR
    biology Review Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR Vivien Yeh , Alice Goode and Boyan B. Bonev * School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; [email protected] (V.Y.); [email protected] (A.G.) * Correspondence: [email protected] Received: 21 October 2020; Accepted: 11 November 2020; Published: 12 November 2020 Simple Summary: Cells, life’s smallest units, are defined within the enclosure of thin, continuous membranes, which confine the molecular machinery required for the life and replication of cells. Crucially, membranes of cells establish and actively maintain distinctly different environments inside cells, including electrical and solute gradients vital to normal cellular functions. Membrane proteins are in charge of transport, electrical polarisation, signalling, membrane remodelling and other important functions. As such, membrane proteins are key drug targets and understanding their structure and function is essential to drug development and cellular control. Membrane proteins have physical characteristics that make such studies very challenging. Nuclear magnetic resonance is one advanced tool that enables structural studies of membrane proteins and their interactions at the atomic level of detail. We discuss the applications of NMR in solution and solid state to membrane protein studies alongside new developments in signal and sensitivity enhancement through dynamic nuclear polarisation. Abstract: Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics.
    [Show full text]
  • Modeling Membrane-Protein Interactions
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 September 2018 doi:10.20944/preprints201809.0055.v1 Peer-reviewed version available at Biomolecules 2018, 8, 120; doi:10.3390/biom8040120 Review Modeling membrane-protein interactions Haleh Alimohamadi and Padmini Rangamani* Department of Mechanical and Aerospace Engineering, University of California San Diego, CA 92093, USA * Correspondence: [email protected]; Tel.: +1-858-534-4734 Abstract: In order to alter and adjust the shape of the membrane, cells harness various mechanisms of curvature generation. Many of these curvature generation mechanisms rely on the interactions between peripheral membrane 1 proteins, integral membrane proteins, and lipids in the bilayer membrane. One of the challenges in modeling these 2 processes is identifying the suitable constitutive relationships that describe the membrane free energy that includes 3 protein distribution and curvature generation capability. Here, we review some of the commonly used continuum elastic 4 membrane models that have been developed for this purpose and discuss their applications. Finally, we address some 5 fundamental challenges that future theoretical methods need to overcome in order to push the boundaries of current model 6 applications. 7 8 Keywords: Plasma membrane; Spontaneous curvature; Helfrich energy; Area difference elastic model; Protein crowding; Deviatoric curvature 9 10 11 1. Introduction 12 The ability of cellular membranes to bend and adapt their configurations is critical for a variety of cellular functions 13 including membrane trafficking processes [1,2], fission [3,4], fusion [5,6], differentiation [7], cell motility [8,9], and signal 14 transduction [10–12]. In order to dynamically reshape the membrane, cells rely on a variety of molecular mechanisms from 15 forces exerted by the cytoskeleton [13–15] and membrane-protein interactions [16–19].
    [Show full text]
  • A New Family of Start Domain Proteins at Membrane Contact Sites Has a Role in ER-PM 2 Sterol Transport
    1 A new family of StART domain proteins at membrane contact sites has a role in ER-PM 2 sterol transport. 3 4 Alberto T Gatta*1, Louise H Wong*1, Yves Y Sere*2, Diana M Calderón-Noreña2, Shamshad 5 Cockcroft3, Anant K Menon2 and Tim P Levine1‡. 6 * contributed equally to this work 7 8 Affiliations: 9 1. Dept of Cell Biology, UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK 10 2. Dept of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA 11 3. Dept of Neuroscience, Physiology and Pharmacology, UCL, Gower Street, London WC1E 6BT, UK 12 13 ‡ to whom correspondence should be addressed: email [email protected]; 14 phone +44/0–20 7608 4027/8 15 16 Major subject area: Cell biology 17 Funding information: AG: Marie Curie ITN “Sphingonet”; LW: Medical Research Council (UK); 18 YS and DMC-N: Qatar National Research Fund. 19 20 21 Competing Interests The authors declare that there are no competing interests. 1 22 ABSTRACT 23 Sterol traffic between the endoplasmic reticulum (ER) and plasma membrane (PM) is a 24 fundamental cellular process that occurs by a poorly understood non-vesicular mechanism. 25 We identified a novel, evolutionarily diverse family of ER membrane proteins with StART-like 26 lipid transfer domains and studied them in yeast. StART-like domains from Ysp2p and its 27 paralog Lam4p specifically bind sterols, and Ysp2p, Lam4p and their homologs Ysp1p and 28 Sip3p target punctate ER-PM contact sites distinct from those occupied by known ER-PM 29 tethers.
    [Show full text]
  • Membrane Proteins Tutorial
    University of Illinois at Urbana-Champaign Beckman Institute for Advanced Science and Technology Theoretical and Computational Biophysics Group Computational Biophysics Workshop Membrane Proteins Tutorial Alek Aksimentiev Marcos Sotomayor David Wells Current editor: Paween Mahinthichaichan November 2016 2 A current version of this tutorial is available at http://www.ks.uiuc.edu/Training/Tutorials/ Join the [email protected] mailing list for additional help. CONTENTS 3 Contents 1 Building a Structural Model of KcsA 6 1.1 Downloading and Viewing the Protein . 6 1.2 Examining the PDB File . 8 1.3 Building the KcsA Tetramer . 9 1.4 Generating PSF and PDB Files for KcsA . 12 1.5 Solvating the Protein (Optional) . 18 2 Placing KcsA in a Membrane 24 2.1 Building a Membrane Patch . 24 2.2 Alignment of Membrane and Protein . 25 2.3 Combination of Membrane and Protein . 26 2.4 Solvation and Ionization . 29 3 Running a Simulation of KcsA 34 3.1 Melting of Lipid Tails . 34 3.2 Minimization and Equilibration with Protein Constrained . 39 3.3 Equilibration with Protein Released . 41 3.4 Production Runs . 43 CONTENTS 4 Introduction This tutorial is designed to guide users of VMD and NAMD through all the steps required to set up a membrane protein system for molecular dynamics simulations. The tutorial assumes that you already have a working knowledge of VMD and NAMD. For the accompanying VMD and NAMD tutorials go to: http://www.ks.uiuc.edu/Training/Tutorials/ This tutorial has been designed specifically for VMD 1.8.6 and NAMD 2.6 but will also work with VMD 1.8.7 and NAMD 2.7.
    [Show full text]
  • Molecular Structure and Function of Myelin Protein P0 in Membrane
    bioRxiv preprint doi: https://doi.org/10.1101/395491; this version posted August 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Molecular structure and function of myelin protein P0 in membrane stacking 2 Arne Raasakka1,2, Salla Ruskamo2, Julia Kowal3,4, Huijong Han2, Anne Baumann1,5, Matti Myllykoski2, 3 Anna Fasano6, Rocco Rossano7, Paolo Riccio7, Jochen Bürck8, Anne S. Ulrich8,9 Henning Stahlberg3 & Petri 4 Kursula1,2,† 5 1Department of Biomedicine, University of Bergen, Bergen, Norway 6 2Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland 7 3Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland 8 4Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, Switzerland 9 5Division of Psychiatry, Haukeland University Hospital, Bergen, Norway 10 6Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy 11 7Department of Sciences, University of Basilicata, Potenza, Italy 12 8Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany 13 9Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany † 14 Corresponding author. E-mail: [email protected] 15 16 17 1 bioRxiv preprint doi: https://doi.org/10.1101/395491; this version posted August 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 Compact myelin forms the basis of nerve insulation essential for higher vertebrates.
    [Show full text]
  • Membrane-Protein Topology
    REVIEWS Membrane-protein topology Gunnar von Heijne Abstract | In the world of membrane proteins, topology defines an important halfway house between the amino-acid sequence and the fully folded three-dimensional structure. Although the concept of membrane-protein topology dates back at least 30 years, recent advances in the field of translocon-mediated membrane-protein assembly, proteome-wide studies of membrane-protein topology and an exponentially growing number of high- resolution membrane-protein structures have given us a deeper understanding of how topology is determined and of how it evolves. Topology In the year 2000, it all seemed so simple: membrane across the membrane in response to drastic changes in A specification of the number proteins of the helix-bundle type were built from long, lipid composition. However, whether such reorientation of transmembrane helices and hydrophobic α-helices that were orientated more or less phenomena are also part of the normal functioning of their in and/or out orientations perpendicularly to the membrane plane1. The topology membrane proteins is less clear. across the membrane in a membrane protein. of a membrane protein was just a description, listing In this review, I summarize the recent developments the segments of the polypeptide chain that form the in our understanding of membrane-protein topology and Fold space transmembrane helices and their orientation relative to structure. I aim to provide a (reasonably) integrated view The abstract space of all the membrane. of membrane-protein biogenesis, which starts from the protein folds. But now, 6 years on and 60 high-resolution membrane- basic process of helix integration into a membrane and ‘Knobs-into-holes’ geometry protein structures later, the picture is not so simple.
    [Show full text]
  • Changes in the Composition of Plasma Membrane Proteins During Differentiation of Embryonic Chick Erythroid Cell
    Proc. NatI. Acad. Sci. USA Vol. 74, No. 3, pp. 1062-1066, March 1977 Cell Biology Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell (red blood cell/development/membrane isolation and characterization/sodium dodecyl sulfate-polyacrylamide gels) LEE-NIEN'L. CHAN Physiology Department, University of Connecticut Health Center, Farmington, Conn. 06032 Communicated by George E. Palade, November 29,1976 ABSTRACT Erythroid cells which are homogeneous with protein composition of plasma membranes from embryonic regard to sta e of maturation are naturally available from the chick erythroid cells at various stages of maturation. Significant circulation of chick embryos at various times of development. This provides a convenient system for examining the changes changes both in quality and in quantity of membrane proteins in plasma membrane protein composition during red cell mat- occur during embryonic erythroid differentiation. uration, Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive char- MATERIALS AND METHODS acterization of the isolated membranes show that they are pure Materials. Fertilized White Leghorn eggs are obtained from and their roteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both the Spafas Co., Norwich, Conn. The eggs are incubated in a qualitative and quantitative changes occur in membrane protein Humidaire Incubator (model no. 50) at 380 (dry bulb) and 290 composition during the early stage of erythroid differentiation. (wet bulb). Red blood cells are collected from the circulation Specific proteins of red cell membrane such as "spectrin" and of the embryos after various times of incubation by cutting open band three proteins are present in low levels in early erythro- the main blood vessels and allowing the blood cells to be blasts but increase in their relative amounts with maturation.
    [Show full text]