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6. Protein targeting and membrane traffic Principles of intracellular organization Signal sequences and targeting signals Membrane translocation mechanisms Tubulo-vesicular membrane traffic Molecular mechanisms of vesicle budding Vesicle transport along microtubules Molecular mechanisms of membrane fusion Neurotransmitter release at neuron synapses 1 (Eukaryote) cells are complex 10 µm Dual-Core Intel® Xeon® processor Bovine pulmonary endothelial cell • 37 x 37 mm • 8.2 108 transistors • 45 nm manufacturing technology • 3 GHz clock speed • 50 x 50 x 5 µm • about 104 different proteins (out of 106 possible) • average protein size 5 nm • typical protein turnover 100 Hz Fast execution of consecutive instructions Able to duplicate itself and to adapt to its environment 2 Microprocessor vs (eukaryote) cells Dual-Core Intel® Xeon® processor Bovine pulmonary endothelial cell • 37 x 37 mm • 8.2 108 transistors • 45 nm manufacturing technology • 3 GHz clock speed • 50 x 50 x 5 µm • about 104 different proteins (out of 106 possible) • average protein size 5 nm • typical protein turnover 100 Hz Communication 10-10 s diffusion limited (DGFP = 90 µm2.s-1 27 s Light speed Hink et al. JBC 275 :17556–17560) Number of components 8.2 108 about 1010 proteins 1 pM < concentration < 100 µM (actin) 1-108 molecules/cell How is cell synchronization achieved ? How do proteins localize properly ? 3 Principles of cellular organization Structural elements : plasma membrane, nucleus, organelles, vesicles, cytoskeletons, ciliae spatial organization of the cell - The range of interaction between macromolecules inside the cell is very short, typically a few nanometers. - Macromolecular complexes (membranes, polymers, complexes) are needed to organize the cell’s interior. - The dynamics of macromolecular complexes is coupled to energy consumption and is able to exert forces and flows sufficient to shape the cell - Proteins are targeted to specific structural elements thanks to targeting signals Roles of the different structural elements : energy production, macromolecule synthesis and degradation, intracellular transport, uptake, secretion, movement, cell division Functional organization of the cell - Membranes delineate cell compartments where different reactions take place. - Membranes and polymers accelerate reactions by reducing the dimensionality of the space where diffusion takes place 4 Localization of main cellular functions plasma membrane transport cell adhesion (toward the ECM and other cells) endocytosis nucleus import and export DNA replication and repair RNA synthesis exocytosis lipid -oxidation citric acid cycle oxidative phosphorylation mitochondria vesicular transport lipid synthesis glycolysis protein synthesis 5 Why intracellular traffic is important ? Diffusion is effective at nm-µm distances self-assembly Above 10 µm, diffusion is very ineffective energy driven assembly and movement Diffusion kinetics : r2(t) = Dt Einstein-Schmoluchowski D = kBT/6phR M-1/3 for globular proteins D (µm2.s-1) Water 3000 + Na 1000 Acetylcholine 200 Insulin (5,7 kDa) 150 GFP (30 kDa) 90 Myosin (250 kDa) 10 r@1msec (µm) 1.7 1 0.5 0.4 0.24 0.027 r@1sec (µm) 55 32 14 12 9 1 Long range organization within the cell : polymers, membrane curvature, mechanical forces self-organization of intracellular compartments Long range organization within tissues : fluid convection, fluid flow, mechanical forces 6 Protein targeting and sorting signals Targeting and sorting signals are amino acid stretches encoded in the primary sequence that define the journey of a given protein in the cell and its final localization. A single protein may contain several targeting and sorting signals. A signal sequence consists of about 20 amino acids at the N-terminal end of the primary sequence of a protein. It allows insertion of the protein in the membrane of an organelle (endoplasmic reticulum, mitochondria...) or translocation of the protein through one or several organelle membranes. When the protein is imported inside the lumen of the organelle, the signal sequence is often cleaved by a specific protease and degraded. Retention signals maintain some proteins in a given compartment, usually by interacting with specific membrane receptors. The steadystate localization of these receptors results from the balance of anterograde and retrograde traffic. Targeting signals are either constitutive (always active) or may be activated by phosphorylation and/or conformational changes. 7 Example of targeting signals Target compartement import in the ER Typical sequence +MMSFVSLLLVGILF WATEAEQLTKCEVFN retention in the ER Import in mitochondria KDEL+MLELRNSIRFFKPATRTLCSSRYLL Import in the nucleus PPKKKRKV Import in peroxysomes SKL Sorting to endosomes ou lysosomes YxxP, ExxLL plasma membrane Acylation (modification by a lipid group) +GSSKSKPK CxxL-, CCxx- plasma membrane membrane proteins and secreted proteins glycosylation at NxS/T sequence Hydrophobic amino acid Negatively charged amino acid Positively charge amino acid 8 Targeting signal characteristics They are specific of a given compartment and well conserved in eukaryotes They allow a reversible interaction with the proteins organizing the intracellular protein transport They are generic and allow targeting of a protein of interest In the absence of any signal, a protein may be targeted to a default localization Example : human cytochrome c oxydase subunit VIII (COX-8) cleavage 10 20 30 40 50 60 MSVLTPLLLR GLTGSARRLP VPRAKIHSLP PEGKLGIMEL AVGLTSCFVT FLLPAGWILS 69 HLETYRRPE Key From TRANSIT 1 CHAIN 26 TOPO_DOM 26 TRANSMEM 37 TOPO_DOM 61 To Length 25 25 69 44 36 11 60 24 69 9 Description Mitochondrion signal sequence Cytochrome c oxidase polypeptide VIII-liver/heart. Mitochondrial matrix transmembrane domain Mitochondrial intermembrane 9 Example of a plasmid used to label mitochondria ‘Living Colors’: set of plasmids encoding fluorescent proteins targeted to specific subcellular compartments Cytomegalovirus promoter (PCMV) Coding sequence : COX-8 sequence signal Ds-Red2 coding sequence MSVLTPLLLR GLTGSARRLP VPRAKRSSKN VIKEFMRFKV RMEGTVNGHE FEIEGEGEGR PYEGHNTVKL KVTKGGPLPF AWDILSPQFQ YGSKVYVKHP ADIPDYKKLS FPEGFKWERV MNFEDGGVVT VTQDSSLQDG CFIYKVKFIG VNFPSDGPVM QKKTMGWEAS TERLYPRDGV LKGEIHKALK LKDGGHYLVE FKSIYMAKKP VQLPGYYYVD SKLDITSHNE DYTIVEQYER TEGRHHLFL Terminator sequence of SV40 virus (SV40polyA) 10 Protein targeting and membrane traffic Principles of intracellular organization Signal sequences and targeting signals Membrane translocation mechanisms Tubulo-vesicular membrane traffic Molecular mechanisms of vesicle budding Vesicle transport along microtubules Molecular mechanisms of membrane fusion Neurotransmitter release at neuron synapses 11 Mechanisms of membrane translocation Example : mitochondrial import Experimental evidence using semi-reconstituted systems proteolysis and SDS-PAGE analysis reveal the existence of a translocation intermediate 12 Protein import pathways into mitochondria Wiedemann, N. et al. J. Biol. Chem. 2004;279:14473-14476 13 Protein translocation into the endoplasmic reticulum ribosome messenger RNA GDP GTP Signal sequence Signal Recognition Particle and ribosomal receptor translocator specific peptidase mature polypeptide for secretion 14 Soluble protein secretion GE Palade and JD Jamieson experiment 1969 pulse : 1 min [3H]-leucine 3 min chase 20 min chase 90 min chase Observation of newly synthesized proteins by autoradiography and electronic microscopy endoplasmic reticulum Golgi apparatus secretion vesicles 15 Plasma membrane recycling endosome late endosome secretory vesicles lysosome Retrograde traffic TGN trans-Golgi medial-Golgi cis-Golgi exocytosis (secretion) endocytosis sorting endosome EGTC endoplasmic reticulum Nucleus 16 Glycosylation of secreted proteins and of transmembrane proteins targeted to the plasma membrane Secreted soluble proteins and transmembrane proteins targeted to the plasma membrane are post-translationally modified by sugar groups (glycosylation). Glycosylation consists of a complex oligosaccharide linked to an asparagine in the sequence NxS/T. Glycosylation sequentially takes place in the endoplasmic reticulum and in Golgi apparatus cisternae. 17 Protein modification in the Golgi apparatus Oligosaccharide attached to an asparagine amino acid Intra-Golgi compartments Ultrastructure of the Golgi apparatus 18 Constitutive and regulated secretion Yeast enzyme secretion Synaptic neurotransmitter secretion Insulin secretion 19 Molecular study of membrane intracellular traffic Identification of proteins involved in membrane traffic : 1. Genetic approach : screening of thermosensitive secretion mutants in Saccharomyces cerevisiae 2. Biochemical approach : in vitro reconstitution of cis-medial intra Golgi transport and purification of soluble factors Study of protein interactions : Interaction between protein partners : co-immunoprecipitation Intracellular localization of proteins : optical microscopy (immunolocalization, FRET) and (immuno) electron microscopy In vitro reconstitution of membrane traffic : In vitro reconstitution of membrane fusion and membrane budding using synthetic vesicles and recombinant proteins In vitro reconstitution of vesicle-cytoskeleton interaction and directed transport Study of regulations : Intracellular specificity of membrane traffic Specific toxins inhibit membrane traffic 20 1. Screening for thermosensitive secretion mutants generated by random mutagenesis in the yeast Saccharomyces cerevisiae R. Scheckman, P. Novick Random generation of mutant cells Thermosensitive mutants 23 °C Permissive temperature 36 °C Restrictive temperature Selection screen enzyme secretion at the restrictive temperature sec mutants (contain a thermosensitive 21 version of a Secx protein) Complementing the molecular defect of thermosensitive mutants secx thermosensitive mutant generated in a TRPD strain constitutive promoter transformation with an expression library random cDNA TRP Selection at the restrictive temperature in the absence of tryptophan TRPD : yeast strain lacking a TRP gene used for tryptophane biosynthesis, which is unable to grow in the absence of tryptophan (auxotroph strain) Most frequent cDNA : wild type gene secx Less frequent cDNA : suppressor genes secy cDNA analysis 22 Sec mutant morphology Sec mutants are classified in five classes according to yeast ultrastructure observed by electron microscopy transport vesicles plasma membrane endoplasmic reticulum Golgi apparatus Golgi apparatus transport vesicles 23 2. Using in vitro reconstituted cell free systems to study vesicular traffic J.E. Rothman principle identification of proteins involved in intra-Golgi membrane traffic Identification of the targets of biochemical inhibitors Cytosol fractionation and reconstitution GTPgS→ARF, coatomer NEM→NSF, SNAP, SNAREs 24 experimental design CHO-15B cells (deficient in N-acetyl glucosamine VSV-G protein transport transferase) infected by intermediates contained the vesicular somatitis in the cis cisternae of the virus (VSV) Golgi stack Wild type CHO cells (contain active N-acetyl glucosamine transferase) Purification of Golgi stacks Cytosol preparation Golgi : donor fraction Golgi : acceptor fraction Cytosol, ATP, UTP UDP-[3H] N-acetyl glucosamine Incubation 37°C Detergent lysis + Immunoprecipitation with anti-VSV-G antibodies Quantitation of [3H] Nacetyl glucosamine associated to VSV-G 25 Biochemical study of protein interaction : co(immuno)précipitation or pull-down techniques 1. Cell extract (membranes solubilized with a detergent) 2. Affinity chromatography Antibodies are often used !!!! Purification with immobilized antibodies Purification of protein complexes Use of magnetic beads reduces contamination with aggregated proteins 3. Identification of the eluted polypeptides by Western blot or mass spectrometry 26 Protein targeting and membrane traffic Principles of intracellular organization Signal sequences and targeting signals Membrane translocation mechanisms Tubulo-vesicular membrane traffic Molecular mechanisms of vesicle budding Vesicle transport along microtubules Molecular mechanisms of membrane fusion Neurotransmitter release at neuron synapses 27 1. Vesicle budding GTP hydrolysis (ARF) Bonifacino et Glick 2004 Cell 116: 153-166 28 Inhibition by the GTPgS or by an ARF mutant unable to hydrolyze its bound GTP Dt 120 min - Dt + GTPgS standard assay Tanigawa et al. 1993 J Cell Biol. 123 : 1365-1371 standard assay + GTPgS 29 Accumulation of coated vesicles in the presence of GTPgS yeast orthologue Sec33 Sec26/27 Sec21 Sec28 Sar1p ARF coatomer COPI coat active in the presence of GTPgS J.E. Rothman & L. Orci 30 Assembly and disassembly of protein coat is coupled to GTP hydrolysis by ARF, a small G protein Example : COP1 (coatomer) Cargo : proteins to be transported Coatomer : a protein complex that binds to the membrane, selects the cargo and deforms the membrane GTPgS ARF (ADP ribosylating factor) : a small G protein GEF : G protein exchange factor Three types of coat exist : clathrin (endocytosis), COPI (secretion, retrograde traffic) and COPII (secretion, anterograde traffic) 31 Model for Membrane Recruitment of Coatomer (A) a composite model of βδ/γζ-COP bound to membrane via two molecules of Arf1-GTP. The γζ-COP/Arf1 crystal structure is colored as is the second molecule of Arf1. The remainder of βδ/γζ-COP, in grey. The Nterminal amphipathic α helices of Arf1 (colored red) are modeled in their expected locations as membrane anchors. (B) Effects of mutations in the Arf1-binding sites of full-length βδ/γζ-COP complex, measured using the pull-down assay. Single mutations reduce but do not abolish Arf1 interaction (lanes 3 and 4), whereas a double mutation binds to Arf1 at background levels (compare lane 5 to lane 1). (C) The effects of single and double mutations in the βδ/γζCOP complex on GTP hydrolysis in the fluorescence assay. 3-O-[N-methyl-anthraniloyl]-GTP + Arf-GAP mant-GTP fluorescence level mant-GDP fluorescence level Xinchao Yu, Marianna Breitman, and Jonathan Goldberg. A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes Cell. 2012 148: 530–542. Partial Model of Coatomer structure A (A) Surface representation of the αβ’-COP triskelion. α-COP and β’-COP subunits are colored in three shades of orange and green, respectively. (B) Known and unknown elements of the αβ’εCOP complex. The speculative element of the diagram is the dimer contact (indicated by the question mark) that brings together two copies of αβ’ε-COP. (C) and (D) Model of an icosahedral COPI cage. This type of structure is formed by clathrin in vitro C Changwook Lee and Jonathan Goldberg. Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII and Clathrin Vesicle Coats Cell. 2010 142 : 123–132. Guanosine-5’(g-thio)triphosphate or GTPgS is a nonhydrolyzable GTP analogue S Other non hydrolyzable analogues exist : GppNHp GDPS is a GDP analogue that cannot be phosphorylated by endogenous nucleoside diphosphate kinase (NDPK) : ATP + GDP → ADP + GTP 34 Arf-GDP structure 1rrf Gln71 Asp67 phosphate Mg2+ a phosphate 5Å GDP 35 Small G protein signalling G proteins have two states (GDP or GTP bound) GEF and GAP controls the transition frequency over two orders of magnitude Specific amino acids (e.g. ArfQ71) are involved in GTP binding or hydrolysis SIGNAL IN GDP/GTP + exchange G protein Exchange Factor (GEF) SIGNAL IN - GTP hydrolysis GTPase activating protein (GAP) 36 Function Vesicle transport and fusion Cell signaling Vesicle formation Actin cytoskeleton regulation Nucleocytoplasmic transport of RNA and proteins Wennerberg K et al. J Cell Sci 2005;118:843-846 2. Vesicle transport along microtubules ATP/GTP hydrolysis (molecular motors) 4’ directed transport Bonifacino et Glick 2004 Cell 116: 153-166 38 The tubulin cytoskeleton Microtubules are tubulin polymers, a protein that binds and hydrolyzes GTP. The cycle of microtubule polymerisation-depolymerisation is coupled to GTP hydrolysis Microtubules are organized radially around the centrosome, also called the MicroTubule Organizing Center (MTOC) which is localized near the nucleus. They allow centripetal or centrifuge organelle transport within the cell. During cell division, the centrosome replicates and organizes chromosome separation by forming a mitotic spindle. Microtubules are rigid and their polymerization exert mechanical forces that allow the centrosome to reach a position determined by the microtubule depolymerization activity along the cell cortex (cell polarization). Untreated cell cellule treated with nocodazole, a microtubule polymerization inhibitor 39 Tubulin and microtubule structure 5 nm Tubulin is incorporated in protofilaments as TaGTPTGTP The tubulin subunit rapidly hydrolyses GTP into GDP TaGTPTGDP protofilaments are instable 40 Microtubule instability CENTROSOME Microtubules minus ends are locked at the centrosome. Microtubule plus end depolymerizes about 100 times faster when it contains GDP tubulin than when it contains GTP tubulin. A GTP cap therefore, favors growth. When it is lost, fast depolymerization occurs. Individual microtubules therefore alternate between a period of slow growth and a period of rapid disassembly, a phenomenon called dynamic instability. - end + end 41 Microtubule dynamics 42 Molecular motors Kinesins are molecular motors that move to the + end of microtubules (anterograde) Dyneins are molecular motors that move to the end of microtubules (retrograde) Further readings : http://www.ncbi.nlm.nih. gov/books/NBK21710/ 43 Höök P , Vallee R B J Cell Sci 2006;119:4369-4371 ©2006 by The Company of Biologists Ltd 3. Membrane fusion GTP hydrolysis (Rab) ATP hydrolysis (NSF) Bonifacino et Glick 2004 Cell 116: 153-166 45 Identification of a protein complex involved in membrane fusion Inhibition of intra Golgi transport by N-ethyl maleimide, a molecule reacting with SH groups purification of NSF (NEM-Sensitive Factor), a soluble ATPase = Sec18 Purification of soluble proteins necessary for NSF binding to membranes identification of SNAP (Soluble NSF Attachment Proteins) = Sec17 Purification of transmembrane proteins necessary for NSF binding to membranes, from brain membranes identification of SNAREs (Soluble NSF Attachment Proteins REceptors) Syntaxin SNAP-25 (25 kDa Synaptosomal Associated Protein) VAMP (vesicle-associated membrane protein) 46 N-ethylmaleimide a-SNAP Soluble NSF Attachment Protein N-ethylmaleimide Sensitive Factor (NSF) Co-immunoprecipitation of a NSF-SNAP-SNAREs complex Söllner et al. 1993 Nature 362: 319-324 syntaxin B g-SNAP syntaxin A a-SNAP SNAP-25 2 Myc-epitope: EQKLISEEDL 3 VAMP/ Synaptobrevin2 SDS-PAGE + mass spectrometry 48 Sutton et al. 1998 Nature 395: 347-353 The SNARE model SNAREs are present on transport vesicles (v-SNAREs) and on target membranes (t-SNARE). The specificity of membrane fusion in ensured by the formation of specific v-t SNARE complexes. SNAREs form a coiled-coil structure consisting of 4 a helices that catalyses membrane fusion (fusion complex). After membrane fusion, NSF and SNAP proteins catalyze SNARE disassembly. The cycle of SNARE complex assembly and assembly is driven by ATP hydrolysis. 49 In vitro reconstitution of membrane fusion Donor NBD-PE + Rhodamine-PE (2%) 50 nm v-SNARE 750/vesicle Acceptor PC, PS, PE 15 x excess t-SNARE 75/vesicle +Triton X-100 Weber et al. 1998 Cell 92 : 759-772 50 Retrograde traffic exocytosis (secretion) 51 endocytosis Protein targeting and membrane traffic Principles of intracellular organization Signal sequences and targeting signals Membrane translocation mechanisms Tubulo-vesicular membrane traffic Molecular mechanisms of vesicle budding Vesicle transport along microtubules Molecular mechanisms of membrane fusion Neurotransmitter release at neuron synapses 52 Vesicular transport summary : energy consumption ATP/GTP hydrolysis (molecular motors) 4’ directed transport GTP hydrolysis (ARF) GTP hydrolysis (Rab) ATP hydrolysis (NSF) Bonifacino et Glick 2004 Cell 116: 153-166 53 At the synapse, neurotransmitters are released by controlled membrane fusion 0.5 msec 54 Pre-assembled SNARE complexes held by synaptotagmin Voltage driven Ca2+ entry Synaptotagmin activation by Ca2+ SNARE-driven membrane fusion 55 Specific neurotoxins cleave neuronal SNARE proteins Syntaxin VAMP SNAP-25 TeNT, tetanus neurotoxin BoNT, botulism neurotoxin G Schiavo (2000 ) Physiological Reviews, 80: 717-766 56 The Botox The BoNT/A toxin blocks the synaptic transmission between nerves and muscles. Intra-muscular injection relaxes muscles of the face. Hyaluronic acid is a natural polymer of the extracellular matrix. Crosslinked hyaluronic acid is intradermically injected to smooth the face surface The effect lasts 3 to 4 months; a single injection costs about 400 €. Botox is often combined with a product to fill wrinkles. 57 G Palade NP 1974 R Scheckman C de Duve L Orci A Claude G Blobel NP 1999 J Rothmann 58 i-bio Seminars Wittinghofer : small G proteins Scheckman : membrane traffic Model for Membrane Recruitment of Coatomer (A) a composite model of βδ/γζ-COP bound to membrane via two molecules of Arf1-GTP. The γζ-COP/Arf1 crystal structure is colored as is the second molecule of Arf1. The remainder of βδ/γζ-COP, in grey. The Nterminal amphipathic α helices of Arf1 (colored red) are modeled in their expected locations as membrane anchors. (B) Effects of mutations in the Arf1-binding sites of full-length βδ/γζ-COP complex, measured using the pull-down assay. Single mutations reduce but do not abolish Arf1 interaction (lanes 3 and 4), whereas a double mutation binds to Arf1 at background levels (compare lane 5 to lane 1). (C) The effects of single and double mutations in the βδ/γζCOP complex on GTP hydrolysis in the fluorescence assay. 3-O-[N-methyl-anthraniloyl]-GTP + Arf-GAP mant-GTP fluorescence level mant-GDP fluorescence level Xinchao Yu, Marianna Breitman, and Jonathan Goldberg. A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes Cell. 2012 148: 530–542. Partial Model of Coatomer structure A (A) Surface representation of the αβ’-COP triskelion. α-COP and β’-COP subunits are colored in three shades of orange and green, respectively. (B) Known and unknown elements of the αβ’εCOP complex. The speculative element of the diagram is the dimer contact (indicated by the question mark) that brings together two copies of αβ’ε-COP. (C) and (D) Model of an icosahedral COPI cage. This type of structure is formed by clathrin in vitro C Changwook Lee and Jonathan Goldberg. Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII and Clathrin Vesicle Coats Cell. 2010 142 : 123–132. Function Vesicle transport and fusion Cell signaling Vesicle formation Actin cytoskeleton regulation Nucleocytoplasmic transport of RNA and proteins Wennerberg K et al. J Cell Sci 2005;118:843-846 N-ethylmaleimide a-SNAP Soluble NSF Attachment Protein N-ethylmaleimide Sensitive Factor (NSF)